Antibodies to ligand growth factors

ABSTRACT

The present invention relates to erbB-2 ligands and functional derivatives thereof which are capable of binding to the erbB-2 oncogene product. The present invention further pertains to anti-ligand molecules capable of recognizing and binding to the erbB-2 ligand molecule and to screening assays for such ligands. The present invention additionally relates to uses for the erbB-2 ligand, the anti-ligand molecules and the screening assays.

The present application is a division of U.S. application Ser. No.08/096,277, filed on Jul. 26, 1993, issued as U.S. Pat. No. 5,578,482,which is a continuation-in-part of U.S. application Ser. No. 07/875,788,filed Apr. 29, 1992, abandoned, which is a continuation-in-part of U.S.application Ser. No. 07/640,497, filed Jan. 14, 1991, abandoned, and acontinuation-in-part of U.S. application Ser. No. 07/917,988, filed Jul.24, 1992, abandoned, which is a continuation-in-part of U.S. applicationSer. No. 07/872,114, filed Apr. 22, 1992, abandoned, which is acontinuation of U.S. application Ser. No. 07/528,438, filed May 25,1990, now abandoned. All of these applications are incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a growth factor which interacts withthe human oncogene erbB-2, and which stimulates as well as inhibits thegrowth of cells overexpressing this oncogene. A ligand is describedwhich is capable of binding to the expression product of the erbB-2oncogene. The present invention additionally relates to anti-ligandmolecules capable of recognizing and binding to the erbB-2 ligandmolecule and to screening assays for such ligands. The present inventionfurther relates to uses for the erbB-2 ligand, the anti-ligand moleculesand the screening assay. Furthermore, the invention relates to a clonedgene capable of expressing the erbB-2 ligand of the present invention.

2. Description of the Related Art

Carcinogenesis is believed to be a multi-step process of alteration ofgenes which are involved in the growth control of cells. A variety ofproto-oncogenes and oncogenes have been implicated in the activation oftumor cells as regulating factors. For example, oncogenic proteinkinases are believed to induce cellular transformation through eitherinappropriate or excessive protein phosphorylation, resulting in theuncontrolled growth of malignant neoplasms. See Wrba, F., et al.,Histopathology, 15:71-76 (1989).

One group of proto-oncogenes encodes cellular growth factors or theirreceptors. The c-erbB-1 gene encodes the epidermal growth factor or itsreceptors. The c-sis gene encodes the B-chain of the platelet-derivedgrowth factor. The c-fms gene encodes a related or identical moleculefor the receptor of the granulocyte-macrophage colony stimulatingfactor. A fourth member of this group of proto-oncogenes, called neu wasidentified in ethylnitrosourea-induced rat neuroblastomas.

The human counterpart of neu, called HER-2/neu or c-erbB-2, has beensequenced and mapped to the chromosomal locus 17q21. See Schneider, P.M., et al., Cancer Research, 49:4968-4971 (Sep. 15, 1989). The HER-2/neuor c-erbB-2 oncogene belongs to the erbB-like oncogene group, and isrelated to, but distinct from the epidermal growth factor receptor(EGFR). The c-erbB-2 oncogene is known to express a 185 kDatransmembrane glycoprotein (p185^(erbB-2)). The expressed protein hasbeen suggested to be a growth factor receptor due to its structuralhomology with EGFR. However, known EGFR ligands, such as EGF or TGFα, donot bind to p185^(erbB-2).

The oncogene has been demonstrated to be implicated in a number of humanadenocarcinomas leading to elevated levels of expression of the p185protein product. For example, the oncogene has been found to beamplified in breast, ovarian, gastric and even lung adenocarcinomas.Furthermore, the amplification of the c-erbB-2 oncogene has been foundin many cases to be a significant, if not the most significant,predictor of both overall survival time and time to relapse in patientssuffering from such forms of cancer. Carcinoma of the breast and ovaryaccount for approximately one-third of all cancers occurring in womenand together are responsible for approximately one-fourth ofcancer-related deaths in females. Significantly, the c-erbB-2 oncogenehas been found to be amplified in 25 to 30% of human primary breastcancers. See Slamon, D., et al., Science, 244, 707-712 (May 12, 1989).

Although ligands for EGFR are known, namely EGF and TGFα, few ligandsfor the oncogene-encoding transmembrane proteins such as erbB-2, ros,etc., have been characterized. Transforming growth factor ligands belongto a family of heat and acid-stable polypeptides which allow cells toassume a transformed morphology and form progressively growing coloniesin anchorage-independent growth assays (DeLarco, et al., Proc. Natl.Acad. Sci. USA, 75:4001-4005 (1978); Moses, et al., Cancer Res.,41:2842-2848 (1981); Ozanne, et al., J. Cell. Physiol., 105:163-180(1980); Roberts, et al., Proc. Natl. Acad. Sci. USA, 77:3494-3498(1980)). The epidermal growth factor receptor (EGFR) and its physiologicligands, epidermal growth factor (EGF) and transforming growth factorα(TGFα), play a prominent role in the growth regulation of many normaland malignant cell types (Carpenter, G., Annu. Rev., Biochem.,56:881-914 (1987)).

One role the EGF receptor system may play in the oncogenic growth ofcells is through autocrine-stimulated growth. If cells express the EGFRand secrete EGF and/or TGFα, then such cells could stimulate their owngrowth. Since some human breast cancer cell lines and tumors expressEGFR (Osborne, et al., J. Clin. Endo. Metab., 55:86-93 (1982);Fitzpatrick, et al., Cancer Res., 44:3442-3447 (1984); Filmus, et al.,Biochem. Biophys. Res. Commun., 128:898-905 (1985); Davidson, et al.,Mol. Endocrinol, 1:216-223 (1987); Sainsbury, et al., Lancet,1:1398-1402 (1987); Perez, et al., Cancer Res. Treat., 4:189-193 (1984))and secrete TGFα (Bates, et al., Cancer Res., 46:1707-1713 (1986);Bates, et al., Mol. Endocrinol, 2:543-555 (1988)), an autocrine growthstimulatory pathway has been proposed in breast cancer (Lippman, et al.,Breast Cancer Res. Treat., 7:59-70 (1986)).

The erbB-2 proto-oncogene amplification has been found in breast,ovarian, gastric, salivary gland, and in non-small cell carcinomas ofthe lung (King, et al., Science, 229:974 (1985); Slamon, et al.,Science, 244:707 (1989); Yokota, et al., Lancet, 1:765 (1986);Fukushige, et al., Mol. Cell. Biol., 6:955 (1986); Semba, et al., Proc.Natl. Acad. Sci. USA, 82:6497 (1985); Weiner, et al., Cancer Res.,50:421 (1990)). Amplification and/or overexpression of the erbB-2protooncogene has been found to correlate with poor prognosis in breast,ovarian and non-small cell lung carcinomas (Slamon, et al., Science,235:177 (1986); Slamon, et al., Science, 244:707 (1989); Guerin, et al.,Oncogene Research, 3:21 (1988); Wright, et al., Cancer Res., 49:2087(1989); Kern, et al., Cancer Res., 50:5184 (1990); DiFiore, et al.,Science, 237:178 (1987)). In addition to these clinical studies, invitro studies strongly suggest that overexpression of the erbB-2transmembrane receptor (p185^(erbB-2)) may have an important role intumor progression (DiFiore, et al., Science, 237:178 (1987); Hudziak, etal., Proc. Natl. Acad. Sci. USA, 84:7159 (1987)).

An autocrine growth stimulatory pathway analogous with that proposed forepidermal growth factor receptor and its ligands may also be employed bya growing list of oncogene encoded transmembrane proteins that havestructure reminiscent of growth factor receptors. This list includes theprotooncogenes neu and its human equivalent erbB-2 or HER2 (Bargmann, etal., Nature, 319:226-229 (1986); Coussens, et al., Science,230:1131-1139 (1985); Yamamoto, et al., Nature, 319:230-234 (1986);c-kit(Yarden, et al., EMBO, 6:341-3351 (1987); ros (Neckameyer, et al.,Mol. Cell. Biol. 6:1478-1486 (1986); met (Park, et al., PNAS,84:6379-6383 (1987); trk (Martin-Zanca, et al., Nature, 319:743-748(1986); and ret (Takahashi, et al., Mol. Cell. Biol., 7:1378-1385(1987)). The erbB-2 and c-kit protooncogenes encode factors that displayremarkable structural homology with EGFR (Yarden, et al., Annu. Rev.Biochem., 57:443-478 (1988). Although erbB-2 and its related oncogeneneu are related to EGFR, these proteins are distinct. For example, knownEGFR ligands such as EGF and TGFα do not bind to erbB-2 receptor. (King,et al., EMBO, 7:1647 (1988); and Stern, et al., EMBO, 7:995 (1988).

If, according to the autocrine growth stimulatory pathway, malignantcells are capable of secreting a potent tumor growth factor in vivo, itis plausible that the growth factor ligand might be detected in bodyfluids, much like human chorionic gonadotropin or α-fetoprotein, andcould be used as a tumor marker and a prognostic variable. Studiessuggest that TGFα activity can be detected in body fluids of cancerpatients and that its presence may provide important informationconcerning the biology of a patient's tumor (Stromberg, et al., J. Cell.Biochem., 32:247-259 (1986); Twardzick, et al., J. Natl. Cancer Inst.,69:793-798 (1982); Sherwin, et al., Cancer Res., 43:403-407 (1983)).

Prior to the present invention, no ligand was known which binds top185^(erbB-2) protein. Thus, a need continues to exist for a ligand forp185^(erbB-2). Such a ligand might be used to counteract the effects ofc-erbB-2 oncogene overexpression in facilitating carcinogenesis.

SUMMARY OF INVENTION

Accordingly, it is an object of the present invention to provide agrowth factor which interacts directly with the erbB-2 oncogene.

It is also an object of the present invention to provide a method forthe isolation and purification of the above-described growth factor.

It is also an object of the present invention to provide a method forstimulating and/or inhibiting the growth of cells which overexpress thehuman oncogene erbB-2.

It is also an object of the present invention to provide a method forgenerally controlling the growth of over-expressing erbB-2 malignantmammalian cells, and, in particular, stimulating the growth of malignantcells at low (physiological) doses.

Accordingly, the above objects and others are provided by anapproximately 30 kDa TGFα-like glycoprotein.

Having obtained the present 30 kDa glycoprotein, in accordance withanother aspect of the present invention, the same is used to inhibit thegrowth of cells which overexpress the c-erbB-2 oncogene.

In accordance with the present invention, the present 30 kDaglycoprotein may be used, by itself, or in conjunction with othermedicinal substances (e.g., toxic moieties or therapeutic agents) toinhibit the growth of any cells which overexpress the c-erbB-2 oncogene.

Generally, the present 30 kDa glycoprotein may be used advantageously toinhibit the growth of adenocarcinoma cells, preferably those of breast,ovarian, gastric and lung tissue which overexpress the erbB-2 oncogene.

In another aspect, the present invention relates to the preparation ofmonoclonal antibodies of gp30, and the use of these monoclonalantibodies to detect the presence of gp30 in patient sera or urine as aprognostic/diagnostic marker for tumor progression.

The present invention thus relates to the use of the present 30 kDaTGFα-like glycoprotein in direct interactions with EGFR andp185^(erbB-2). Hence, in another aspect, the present invention providesconjugates of the 30 kDa glycoprotein ligand with either EGFR orp185^(erbB-2). In still another aspect, the present invention providesdiagnostic and therapeutic methods using these conjugates. Further, thepresent invention provides a diagnostic test kit using the presentconjugates.

The present invention relates to an approximately 75 kilodalton growthfactor ligand or functional derivative thereof which bind specificallyto an erbB-2 oncogene product (p185^(erbB-2)) but fail to recognize andbind to an homologous transmembrane protein, i.e., epidermal growthfactor receptor. Methods of obtaining the purified ligands of thepresent invention are also included in the present invention.

The invention additionally pertains to anti-ligand molecules such asantibodies or fragments of antibodies and blocking peptides which bindto the erbB-2 ligand of the present invention. A method to detect thepresence of cells which express the erbB-2 ligand with these anti-ligandmolecules is also disclosed. A further aspect of the invention involvesthe use of the erbB-2 ligand to detect cells expressing the erbB-2oncogene product, p185^(erbB-2).

The invention further pertains to a recombinant DNA molecule coding fora gene which is capable of expressing the erbB-2 ligand of the presentinvention and to host cells which contain such a recombinant DNAmolecule.

The invention is also directed to a method for treating a number ofcancers associated with the erbB-2 oncogene product overexpressionincluding breast, ovarian, gastric, lung, prostate, salivary gland andthyroid carcinomas.

Lupu, et al., Science, 249:1552-1555 (1990) identified an approximately30 kilodalton (kDa) glycoprotein (gp30) which is similar to TGFα in itsability to bind to the EGFR, phosphorylate EGFR, and induce colonyformation. Direct binding of the gp30 to p185^(erbB-2) was confirmed bybinding competition experiments, suggesting that gp30 is a ligand forp185^(erbB-2). Thus, Lupu, et al., identified and characterized a 30 kDaligand that binds to erbB-2 receptor with high affinity and to EGFR withlower affinity.

Prior to the present invention, no ligand was known which binds to theerbB-2 oncogene product (p185^(erbB-2)) but fails to react with EGFR.Such a ligand will be important for understanding the function ofp185^(erbB-2) and may be a potential therapeutic and diagnostic targetfor neoplasia.

Ligands for erbB-2 are of extreme interest. They may directly modulatethe growth of cancer cells expressing this receptor. They may beconjugated or otherwise coupled to a variety of toxins, drugs andisotopes, for example, to target these therapies to cancer for increasedtherapeutic efficacy or for imaging purposes. In addition, briefstimulation of a cancer by the ligand may be combined with subsequentchemotherapy to increase the responsivity of the cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the isolation of the present 30 kDa growth factor.Portion A illustrates the use of low affinity heparin chromatography,while portion B illustrates the use of reversed-phase chromatography.

FIG. 2 illustrates the detection of phosphorylated proteins in SK-Br-3cells.

FIG. 3 illustrates the detection of phosphorylated proteins inMDA-MB-453 cells.

FIG. 4A illustrates the phosphorylation of p185^(erbB-2) protein inintact CHO/DHFR and FIG. 4B illustrates the phosphorylation ofp185^(erbB-2) protein in intact CHO/erbB-2 cells.

FIG. 5 illustrates a p185^(erbB-2) receptor competition assay in SK-Br-3cells.

FIG. 6 illustrates the inhibition of p185^(erbB-2) cross-linking with4D5 antibody by gp30.

FIG. 7 shows SDS-polyacrylamide gel electrophoresis of samples elutedfrom an affinity column coupled to p185^(erbB-2) extracellular domain.

FIG. 8A shows detection of phosphorylated proteins from cells incubatedin the presence of gp30 or p75. Control media did not contain theseligand molecules. FIG, 8B shows the specificity of the tyrosinephosphorylation. FIG. 8C shows that p75 does not induce phosphorylationof the EFGR in MDA-MB-468 cells.

FIG. 9 depicts the effect of p75 on the growth of human breast cancercells.

FIG. 10 shows the effect of p75 on the soft agar colony formation ofhuman breast cancer cells.

FIG. 11 shows the effect of soluble p185^(erbB-2) extracellular domainon the soft agar colony formation of human breast cancer cells.

FIG. 12 illustrates the effect of gp30 on soft agar colony formation ofSK-Br-3 cells.

FIG. 13 illustrates the effect of gp30 on soft agar colony formation ofMDA-468 cells.

FIG. 14 illustrates the effect of gp30 on soft agar colony formation ofMCF-7 cells.

FIG. 15 illustrates the effect of EGF on soft agar colony formation ofSK-Br-3 cells.

FIG. 16 illustrates the effect of EGF on soft agar colony formation ofMDA-MB-468 cells.

FIG. 17A-B show separation of tryptic digested gp30 by C18-ReversedPhase chromatography and amino acid composition of derived peptides.FIG. 17A shows tryptic digestion/C18 chromatography; FIG. 17B shows thesequences obtained from isolated marked peaks.

FIG. 18A shows the full cDNA sequence of gp30/α1 and FIG. 18B shows afull cDNA sequence β1, FIG. 18C shows the sequence of four differentgp30 isoforms and FIG. 18D demonstrates four different gp30 isoforms inbreast cancer cells by RNase protection assay.

FIG. 19 shows induction of erbB-2 tyrosine phosphorylation using mediafrom MCF-7/ligand expressing cells.

FIG. 20A show binding and covalent cross-linking of radiolabeled gp30 top185^(erbB-2). FIG. 20B shows that the cross-linking of gp30 top185^(erbD-2) is in a dose-dependent fashion.

FIG. 21 shows Western Blot analysis of partially purified ligand.

FIG. 22A shows immunostaining with a polyclonal α1 antibody for theerbB-2 ligands in breast cancer tissue. FIG. 22B shows immunostainingwith a monoclonal antibody 7B3-strait hybridoma supernatant in breastcancer tissue. FIG. 22C represents phase staining. FIG. 22D showsimmunostaining with a monoclonal antibody 10F10-strait hybridomasupernatant in breast cancer tissue. FIG. 22E shows immunostaining witha monoclonal antibody 10F10-strait hybridoma supernatant in the presenceof a blocking peptide. FIG. 22F represents phase staining.

FIG. 23A-C shows generation of a specific erbB-2 ligand PCR product frombreast cancer specimens. FIG. 23A shows the primer derived from α1sequence. FIG. 23B shows the sequence amplified. FIG. 23C shows therestriction digestion of the PCR products.

FIG. 24 shows gp30 effects on SKBR-3 cell invasion and migration in theBoyden chamber.

FIG. 25 shows the effects of estradiol and gp30 on estrogen andprogesterone receptor expression.

FIG. 26 shows erbB-2 receptor binding in BT-474 cells cultured in thepresence of erbB-2 ligands.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated upon the discovery that hormonedependent or independent breast cancer cells secrete growth factors,including an insulin-like growth factor I activity, insulin-like growthfactor II, transforming growth factor alpha platelet-derived growthfactor, members of the FGF family and erbB-2 ligands. Secretion of someof these factors is stimulated by estradiol, and antiestrogens act bydecreasing the secretion of these growth factors in hormone dependentbreast cancers but not in other tumors.

In fact, a variety of strategies which either block the secretion ofthese growth factors in vitro can interfere with the growth of humanbreast cancer cells. Such strategies may include the use of anti-growthfactor antibodies, anti-growth factor receptor antibodies, syntheticpeptides, drugs which interfere with the ligand-receptor interaction,inhibitory ligands, stable transfection of breast cancer cells withantisense genes or specific growth factor receptors or short termtreatment with antisense oligonucleotides to growth factor receptors.

Generally, the present invention provides ligands for p185^(erbB-2),which are capable of generally controlling the growth of erbB-2overexpressing (o/e) cells when applied thereto, and, in particular,either inhibiting or stimulating the growth of o/e erbB-2 cells whenapplied thereto.

In the description that follows, a number of terms used in the field ofligand-growth factor receptor interactions and recombinant DNAtechnology are extensively utilized. In order to provide a clearer andconsistent understanding of the specification and claims, including thescope to be given such terms, the following definitions are provided.

Mutant. As used herein, the term "mutant" is meant to includederivatives of an erbB-2 ligand in which the amino acid sequence of theprotein has been modified in a manner resulting from addition,substitution, insertion or deletion of one or more amino acids in orfrom the wild type protein. By a "biologically active mutant" of aerbB-2 ligand is meant a mutant of the ligand which retains all or someof the biological activity possessed by the ligand, particularly thereceptor binding activity, and most particularly the stimulation ofp185^(erbB-2) autophosphorylation. Mutation may also be used as ageneral term to denote the modification of any DNA or RNA sequence byaddition, substitution, insertion or deletion of one or more nucleotideswithin that sequence.

Functional Derivative. By a "functional derivative" of the erbB-2 ligandof the invention is meant a ligand that possesses a biological activitywhich is substantially similar to the ligand from which the derivativeis derived. By "substantially similar" is meant a biological activitywhich is qualitatively similar but quantitatively different from anactivity possessed by a normal erbB-2 ligand. By the phrase "abiological activity which is qualitatively similar" is meant a ligandwhich more or less retains the biological activity of the natural erbB-2ligand. For example, a functional derivative of the erbB-2 ligandretains the p185^(erbB-2) receptor binding activity, and preferablyretains the ability to stimulate autophosphorylation of p185^(erbB-2).The term "functional derivative" is intended to include biologicallyactive "mutants," "fragments," and "variants," of the erbB-2 ligand.

Fragment. A "fragment" of the erbB-2 ligand is meant to refer to aprotein molecule which contains a portion of the complete amino acidsequence of the wild type ligand. By a "biologically active fragment" ofa ligand is meant a fragment of the erbB-2 ligand which retains all orsome of the biological activity possessed by the ligand. For example, ifthe fragment retains some or all of the receptor binding activity, thensuch fragment is said to be a biologically active fragment of erbB-2ligand.

Variant. A "variant" of the erbB-2 ligand is meant to refer to a ligandsubstantially similar in structure and biological activity to either thenative erbB-2 ligand or to a fragment thereof, but not identical to suchmolecule or fragment thereof. A variant is not necessarily derived fromthe native molecule and may be obtained from any of a variety of similaror different cell lines. The term "variant" is also intended to includegenetic alleles. Thus, provided that two erbB-2 ligands possess asimilar structure and biological activity, they are considered variantsas that term is used herein even if the composition or secondary,tertiary, or quaternary structure of one of the ligands is not identicalto that found in the other.

Generally, erbB-2 ligand variants will have amino acid sequences thatcorrespond to each other. One amino acid sequence "corresponds" toanother amino acid sequence if at least 75% of the amino acid positionsin the first sequence are occupied by the same amino acid residues inthe second sequence. Preferably 90% of the amino acid positions areidentical, and most preferably 95% of the amino acid positions areidentical. Alternatively, two amino acid sequences are considered tocorrespond to each other if the differences between the two sequencesinvolve only conservative substitutions.

"Conservative amino acid substitutions" are the substitution of oneamino acid residue in a sequence by another residue of similarproperties, such that the secondary and tertiary structure of theresultant peptides are substantially the same. Conservative amino acidsubstitutions occur when an amino acid has substantially the same chargeas the amino acid for which it is substituted and the substitution hasno significant effect on the local conformation of the protein. Aminoacid pairs which may be conservatively substituted for one another arewell-known to those of ordinary skill in the art.

As used herein, the term "variant" is meant to include polypeptides ornucleic acids encoding polypeptides that are substantially homologous.Two amino acid sequences are "substantially homologous" when at leastabout 90% of the amino acids match over the defined length of the aminoacid sequences, preferably a match of at least about 92%, morepreferably a match of at least about 95%. Preferred variants of erbB-2ligands contain amino acid sequences that differ from the sequence ofother erbB-2 ligands by 25 or fewer amino acid residues, morepreferably, 18 or fewer residues, even more preferably about 12 or fewerresidues and most preferably about 10 or fewer residues.

Two DNA sequences are "substantially homologous" when at least about 85%(preferably at least about 90%, and most preferably at least about 95%)of the nucleotides match over the defined length of the DNA sequences.Sequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See e.g.,Maniatis et al., supra; DNA Cloning, vols. 1 and II supra; Nucleic AcidHybridization, supra. DNA sequences encoding erbB-2 ligands aresubstantially homologous if they hybridize under stringent hybridizationconditions as defined in International Patent Publication WO 92/20798(incorporated herein by reference).

One DNA sequence "corresponds" to another DNA sequence if the twosequences encode the same amino acid sequence.

erbB-2 ligand. The term "erbB-2 ligand" is meant to refer to a proteinmolecule which is capable of specifically binding to an erbB-2 oncogeneproduct (p185^(erbB-2)). Preferred erbB-2 ligands have one or more ofthe biological activities of the polypeptides encoded by the DNAsequences in FIGS. 18A and B. In particular, preferred erbB-2 ligandsare capable of activating p185^(erbB-2;) most particularly, preferrederbB-2 ligands stimulate autophosphorylation of p185^(erbB-2) Mostpreferred erbB-2 ligands are those that fail to bind to the epidermalgrowth factor receptor. As used herein, the term "erbB-2 ligand" ismeant to include any functional derivative of the erbB-2 ligand of thepresent invention. The erbB-2 ligands of the present invention may bindother protooncogene encoded transmembrane proteins such as c-kit, neu,ros, etc., and thus the term "erbB-2 ligand" is not limited to proteinmolecules which only bind the erbB-2 oncogene product. Binding of theerbB-2 ligand molecules of the present invention may induce cellularresponses of cells which express such other protooncogenes and thus maybe used to treat and diagnose patients that have malignant cells whichexpress these other protooncogenes.

A composition comprising a selected polypeptide component is"substantially pure" when the polypeptide component makes up at leastabout 75% by weight of the combined weight of polypeptide components inthe composition. Preferably, the selected component comprises at leastabout 90% by weight of the combined weight, most preferably at leastabout 99% by weight of the combined weight. In the case of a compositioncomprising a selected biologically active protein, which issubstantially free of contaminating proteins, it is sometimes preferredthat the composition having the activity of the protein of interestcontain species with only a single molecular weight (i.e., a"homogeneous" composition).

A. Ligands of the erbB-2 Transmembrane Protein

The human c-erbB-2 oncogene encodes a 185 kDa transmembrane glycoproteinhaving protein kinase activity. This glycoprotein, known asp185^(erbB-2), shows extensive structural similarity with the p170epidermal growth factor receptor (EGFR) and is therefore thought to begrowth factor receptor. However, neither EGF nor TGFα, the normalligands for the EGFR, interact directly with p185^(erbB-2). In fact, noligand for this glycoprotein has been described prior to this invention.It would be extremely desirable to find a ligand for this 185 kDaglycoprotein, inasmuch as erbB-2 oncogene is amplified in manyadenocarcinomas and is over expressed in nearly 30% of human breastcancer patients. Additionally, it is known that p185^(erbB-2) isnecessary for the maintenance of the malignant phenotype of cellstransformed by the oncogene.

In accordance with the present invention, it has been surprisinglydiscovered that a number of structurally distinct polypeptides functionas ligands for p185^(erbB-2) These ligands include polypeptides of about20-26 kDa (which are glycosylated to form ligands of 30-45 kDa apparentmolecular weight) and also include polypeptides of about 75 kDa whichare not glycosylated. These ligands share the properties of specificallybinding to p185^(erbB-2) and inducing autophosphorylation thereof. Theligands differ in structure and some other biological activities. All ofthe polypeptides which specifically induce autophosphorylation ofp185^(erbB-2) are termed "erbB-2 ligands" herein. The low molecularweight glycosylated species of erbB-2 ligands are variously describedherein by the terms "heregulin", "gp30", "30 kDa growth factor", "30 kDaligand", or "TGFα-like polypeptide". The higher molecular weight speciesis additionally identified as "p75".

An approximately 30 kDa growth factor (gp30) which is secreted from theestrogen receptor negative cell line MDA-MB-231 is effective as a ligandfor p185^(erbB-2) glycoprotein. The 30 kDa glycoprotein of the presentinvention also exhibits some TGFα-like activity. For example, thepresent 30 kDa glycoprotein binds to EGFR, is capable of phosphorylatingEGFR as well as inducing NRK colony formation, although with a loweraffinity than either EGF or TGFα. This is quite surprising inasmuch asthe present 30 kDa growth factor is distinct from the normal 16-18 kDaprecursor for TGFα or mature TGFα as shown by peptide mapping of thetranslated proteins. The 30 kDa glycoprotein was observed, unlike EGFand TGFα, to bind to heparin-sepharose, and can be purified to apparenthomogeneity by heparin affinity chromatography and subsequent reversedphase chromatography. The heparin binding ability of gp30 is a novel andsurprising finding for a growth factor from the EGF family.

The gp30 glycoprotein binds to epidermal growth factor receptor (EGFR)and has TGFα-related properties. In addition, purified gp30 stimulatesphosphorylation of p185^(erbB-2) in cells that overexpress erbB-2, incontrast with TGFα and EGF which do not interact with p185^(erbB-2).Surprisingly, gp30 inhibits cell growth in all cells that overexpressederbB-2 (Lupu, et al., Science, 249:1552 (1990)). A monoclonal antibody(4D5) against the extracellular domain of p185^(erbB-2) (Hudziak, etal., Molec. Cell. Biol., 9:1165 (1989)) was able to compete with gp30for binding to p185^(erbB-2), indicating that the gp30 ligand recognizesand binds to the 4D5 binding site.

However, in accordance with another aspect of the present invention, ithas been surprisingly discovered that very low concentrations of gp30have a stimulatory effect on cells as evidenced by both standardmitogenesis assays and clonogenic assays. By contrast, at higherconcentrations, the ligand is growth inhibitory in both assays.

In accordance with the present invention, it has been found that gp30competes for binding with antibodies directed against erbB-2 whichinhibit growth. Further, it has also been found that the gp30 ligand atlow concentrations is capable of reversing antibody-induced growthinhibition. Additionally, the gp30 ligand can overcome inhibitoryeffects seen in cells which overexpress erbB-2 protein which are inducedby extracellular domain fragments of the erbB-2 receptor, whichindicates a specific pathway of action for the gp30 ligands mediated forinteraction with erbB-2.

Due to the ability of the gp30 ligand to compete with monoclonalantibodies for binding the erbB-2, the present invention also provides aradioreceptor assay in which erbB-2 ligands can be identified by theirability to displace radiolabeled antibodies from binding to erbB-2. Thepresent invention thus provides an affinity chromatography purificationtechnique using soluble erbB-2 extracellular domain.

Generally, the 30 kDa glycoprotein can be immunoprecipitated by ananti-TGFα polyclonal antibody and exhibits some TGFα-like biologicalactivity when assayed by EGF radioreceptor assay and NRK and AIN4T cellcolony formation assays. The 30 kDa growth factor also stimulatesautophosphorylation of the EGF receptor, although less efficiently thanmature 6 kDa TFGα or EGF.

Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitrorevealed a precursor of 22 kDa in contrast to the 16-18 kDa precursorfor mature TGFα. Furthermore, in vitro translation of total mRNA fromMDA-MB-231 cells confirmed these observations. Biochemicalcharacterization of the 30kDa TGFα-like protein was obtained byV8-protease digestion of the de-glycosylated polypeptides and translatedproducts. Peptide mapping of the V8-digested, immunoprecipitatedmaterial suggests an amino acid sequence distinct from TGFα. Hence, the30 kDa polypeptide, while related to the EGF/TGFα family, is encoded bya different gene and is not a post-translation modification of matureTGFα.

The 30 Kd glycoprotein of the present invention is well-characterizedby:

1) being a heparin binding growth factor;

2) being capable of strongly binding to erbB-2;

3) being capable of induce tyrosine phosphorylation of p185^(erbB-2) ;

4) being capable of inducing internalization of the erbB-2 receptor;

5) being capable of stimulate growth of overexpressing erbB-2 cells atlow concentrations;

6) being capable of inhibiting growth of erbB-2 overexpressing cells athigh concentrations;

7) being capable of competing with specific erbB-2 monoclonalantibodies, which antibodies are capable of inducing growth inhibitionof erbB-2 overexpressing cells; and

8) being capable of inducing differentiation of overexpressing erbB-2cells at high concentrations;

In accordance with the present invention, as described above, it hasalso has been discovered that gp30, as well as EGF and TGFα induce cellproliferation of cells such as NRK cells and immortalized human breastepithelial A1N4 cells. Hence, all three ligands have stimulatoryactivity on cells containing high amounts of EGFR, because of theirability to interact with EGFR.

Accordingly, the 30 kDa glycoprotein of the present invention may befurther characterized by:

1) being capable of weakly binding to EGF receptor;

2) exhibiting cross-reactivity to antibodies to TGFα;

3) being capable of cleavage by elastase; and

4) being capable of stimulating transforming activity in normal ratkidney (NRK) cells.

The erbB-2 ligands of the present invention also includes a 75kilodalton protein (p75), although the invention is intended to includeany functional derivatives of this factor. Substantially purified p75ligand competes for p185^(erbB-2) binding with monoclonal antibodiesthat bind to p185^(erbB-2) so that proliferation of erbB-2overexpressing cells is inhibited, such as monoclonal antibody 4D5(Hudziak, et al., Mol. Cell. Biol., 9:1165 (1989)), and p75 inducesphosphorylation of p185^(erbB-2). In cell growth assays, cellproliferation and colony formation of cell lines overexpressing erbB-2were inhibited with high concentration of p75. Furthermore, p75 canreverse the antiproliferative effect of soluble erbB-2 extracellulardomain (ECD).

The 75 kDa erbB-2 ligand of the present invention is extremely importantbecause of the specificity for p185^(erbB-2). Surprisingly, this erbB-2ligand does not recognize or bind to EGFR, a highly homologous receptorto p185^(erbB-2). This characteristic allows the design of diagnosticand therapeutic agents specifically directed against carcinoma cellswhich overexpress erbB-2.

B. Identification of erbB-2 Ligands

Identification of erbB-2 ligands of the present invention can beaccomplished by using a radioreceptor assay to screen conditioned mediafrom a number of cells. Any cell type may be used in a screen to isolateligand-producing cells. Preferably, erbB-2 overexpressing cells areused.

The radioreceptor assay, according to the present invention, utilizes alabeled antibody which binds to the extracellular domain ofp185^(erbB-2). Antibodies directed against the erbB-2 receptorextracellular domain are well known. The preferred antibodies foridentifying erbB-2 ligands of the present invention are 4D5 (Hudziak, etal., Mol. Cell. Biol., 9:1165 (1989)), which may be obtained fromGenentech, Calif.

The antibody 4D5 binds to the same binding site of the extracellulardomain of p185^(erbB-2) such that the erbB-2 ligand of the presentinvention is inhibited from binding the receptor in the presence ofthese antibodies. Thus, these antibodies can be used in competitivebinding assays to identify cell lines that produce the erbB-2 ligand ofthe invention. One of skill in the art will appreciate that otherantibodies which recognize different binding sites or epitopes on theextracellular domain can be generated by well known techniques toidentify a number of ligands not previously described. Thus, use ofdifferent antibodies which bind distinct locations on the extracellulardomain of p185^(erbB-2) may provide for the isolation of unique ligands.

In the assay of the present invention, conditioned media is preparedaccording to commonly employed procedures. For instance, media from acell culture is cleared from cells and concentrated 100 fold in anAmicon ultrafiltration unit (Yarden, et al., Proc. Natl. Acad. Sci.,86:3179-3183 (1989); Lupu, et al., Biochemistry, 31:7330-7340(1992); andBates, et al., Cancer Res. 46:1707-1713 (1986).

Competitive binding of the labeled antibody to p185^(erbB-2) in thepresence of conditioned media provides a method for detecting cellswhich produce ligands. In this manner, the ligand in the conditionedmedia will compete with the labeled antibody for binding to thep185^(erbB-2) protein. Monitoring the amount of label bound to erbB-2protein is determinative of the presence of ligand. For example,decrease in label attached to the erbB-2 receptor indicates the presenceof ligand.

In order to determine whether gp30 bonded specifically to p185^(erbB-2),p185^(erbB-2) binding competition assays were performed. Since iodinatedgp30 was not available, an iodinated anti-erbB-2 (4D5) that inducedsimilar biological responses to gp30 in cells with erbB-2 overexpressionwas used. Iodinated 4D5 MAb was used for the receptor bindingexperiments, in the presence of increasing concentrations of gp30. Thegp30 displaced 4D5 binding to p185^(erbB-2) in intact SK-Br-3 andMDA-MB-453 cells clearly indicating that gp30 binds to the receptor. Ina control experiment, the binding to erbB-2 of an iodinated antibodythat does not show anti-proliferative effects was not altered by gp30.The gp30 binding activity was not inhibited by excess concentrations ofEGF or TGFα.

In order to verify that the receptor competition was specific, iodinated4D5 was covalently cross-linked to p185^(erbB-2) in the presence orabsence of gp30. The complex was immunoprecipitated with an antibody tothe COOH-terminal domain of p185^(erbB-2) and analyzed bySDS-polyacrylamide gel electrophoresis (PAGE). The autoradiogram showeda specific high molecular weight 4D5 binding site. Cross-linking ofp185^(erbB-2) and iodinated 4D5 was blocked in the presence of gp30 .Blocking was not observed in the presence of EGF.

Clearly, gp30 secreted by the MDA-MB-231 breast cancer cell line is aligand for p185^(erbB-2). Moreover, gp30 also is capable of stimulatingp185^(erbB-2) and EGFR phosphorylation. Hence, gp30 can interactdirectly and independently with p185^(erbB-2) and EGFR, and isconsidered to exhibit auto-stimulatory properties.

The erbB-2 ligands of this invention can be identified or detected byother procedures, including competitive binding studies with EGF (forgp30 species), direct binding to p185^(erbB-2) or its ECD,autophosphorylation assays with p185^(erbB-2) cells, or binding ofantibodies to erbB-2 ligands. These procedures are described in detailbelow, especially in Examples 3, 8-12 and 14-19.

C. Purification of erbB-2 Ligand

In accordance with this invention, erbB-2 ligand can be isolated from acell producing the ligand. Any cell that produces the ligand may be usedas a starting material according to the methods described in thisinvention. Lupu, et al., Science, 249:1552-1555 (1990) reported theidentification and purification of a 30 kilodalton (kDa) growth factorsecreted by MDA-MB-231 human breast cancer cells. This glycoprotein(gp30) was purified to apparent homogeneity by sequential low affinityheparin-sepharose chromatography and by reversed phase chromatography.Preferably, SK-Br-3 is used to isolate the 75 kDa erbB-2 ligand of thepresent invention. This strain is well known to those of skill in theart and is deposited with the American Type Culture Collection,Rockville, Md., 20852 USA (accession number ATCC HTB 30). However, anycell which is found to contain the erbB-2 ligand or functionalderivatives thereof can be used to isolate and purify such a factor fromthe cell and/or its culture medium. The ligands of the present inventioncan, for example, be isolated from a host cell which expresses arecombinant ligand.

The ligand of the present invention is likely to be excreted from thecell. Accordingly, the ligand will normally be purified from the culturemedia. However, cellular extracts may serve as a source from which topurify the ligand of the present invention. Typically, the cellsproducing the desired ligand are grown in media conducive to cellgrowth. The cells are removed and the desired ligand is purified fromthe media.

The ligands of the present invention can be extracted and purified fromthe culture media or cell by using known protein purification techniquescommonly employed, such as extraction, precipitation, ion exchangechromatography, affinity chromatography, gel filtration and the like.The most preferred methods to isolate the erbB-2 ligand of the presentinvention are by affinity chromatography using the erbB-2 receptorextracellular domain bound to a column matrix or by heparinchromatography.

As will be apparent to those of skill in the art, extracellular domainobtained from a natural host producing the protein can be used to purifythe ligand of the present invention. For example, SK-Br-3 cells havebeen used to purify the extracellular domain of p185^(erbB-2) (Alper, etal., Cell Growth and Differentiation 1:591-599 (1990)). Alternatively,purified recombinant extracellular domain of p185^(erbB-2) can be usedin affinity chromatography to obtain the erbB-2 ligand of the presentinvention. It will be appreciated that the whole p185^(erbB-2) receptorprotein or portions of such a protein may be used to purify the erbB-2ligand according to the present invention, provided that the proteinbound to the column matrix contains the desired erbB-2 ligand bindingsite of the extracellular domain. Yamamoto, et al., Nature, 319:230-234(1986) describes cloning and expression of the full length p185^(erbB-2)gene. A plasmid containing the erbB-2 receptor gene can be obtained fromthe American Type Culture Collection, Rockville, Md. (Accession No. ATCC57584).

Using an affinity chromatography purification procedure, erbB-2 ligandwas substantially purified from the cellular media. As used herein, theterm "substantially pure" or "substantially purified" is meant todescribe a ligand which is substantially free of any compound normallyassociated with the protein in its natural state, i.e., substantiallyfree of contaminating protein and carbohydrate components. The term isfurther meant to describe a ligand of the present invention which ishomogeneous by one or more purity or homogeneity characteristics used bythose of skill in the art. For example, substantially pure ligandproteins will show constant and reproducible characteristics withinstandard experimental deviations for parameters such as the following:molecular weight, chromatographic techniques, and such other parameters.The term, however, is not meant to exclude artificial or syntheticmixtures of the erbB-2 ligand with other compounds. The term is also notmeant to exclude the presence of minor impurities which do not interferewith the biological activity of the enzyme, and which may be present,for example, due to incomplete purification.

D. Cloning erbB-2 Ligand Genes

Any of a variety of procedures may be used to clone the erbB-2 ligandgenes of the present invention. One such method entails analyzing ashuttle vector library of DNA inserts (derived from a cell whichexpresses the erbB-2 ligand) for the presence of an insert whichcontains the ligand gene. Such an analysis may be conducted bytransfecting cells with the vector and then assaying for expression ofthe ligand binding activity. The preferred method for cloning thesegenes entails determining the amino acid sequence of the erbB-2 ligandprotein. Usually this task will be accomplished by purifying the desiredligand protein and analyzing it with automated sequencers.Alternatively, each protein may be fragmented as with cyanogen bromide,or with proteases such as papain, chymotrypsin or trypsin (Oike, Y., etal., J. Biol. Chem., 257:9751-9758 (1982); Liu, C., et al., Int. J.Pept. Protein Res., 21:209-215 (1983)). Although it is possible todetermine the entire amino acid sequence of these proteins, it ispreferable to determine the sequence of peptide fragments of thesemolecules. Once one or more suitable peptide fragments have beensequenced, the DNA sequences capable of encoding them are examined andone or more suitable oligodeoxyribonucleotides which encode a fragmentof the desired erbB-2 ligand sequence are identified. Because thegenetic code is degenerate, more than one codon may be used to encode aparticular amino acid (Watson, J. D., In: Molecular Biology of the Gene,3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977), pp. 356-357).The oligonucleotides were synthesized at LCRC and at Biosynthesis, Tx.The peptide fragments are analyzed to identify sequences of amino acidswhich may be encoded by oligonucleotides having the lowest degree ofdegeneracy. This is preferably accomplished by identifying sequencesthat contain amino acids which are encoded by only a single codon.Although occasionally such amino acid sequences may be encoded by only asingle oligonucleotide, frequently the amino acid sequence can beencoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of the set contain oligonucleotides which arecapable of encoding the peptide fragment and, thus, potentially containthe same nucleotide sequence as the gene which encodes the peptidefragment, only one member of the set contains a nucleotide sequence thatis identical to the nucleotide sequence of this gene. Because thismember is present within the set, and is capable of hybridizing to DNAeven in the presence of the other members of the set, it is possible toemploy the unfractionated set of oligonucleotides in the same manner inwhich one would employ a single oligonucleotide to clone the gene thatencodes the peptide. Alternatively, a suitable oligonucleotide that iscapable of encoding a fragment of an erbB-2 ligand may be identified inthe erbB-2 ligand sequence provided herein in FIG. 17B, or theoligonucleotide of FIGS. 18A and B or 23A and B may be used.

In a manner exactly analogous to that described above, one may employ anoligonucleotide (or set of oligonucleotides) which have a nucleotidesequence that is complementary to the oligonucleotide sequence or set ofsequences that is capable of encoding the peptide fragment.

A suitable oligonucleotide, or set of oligonucleotides which is capableof encoding a fragment of the desired erbB-2 ligand gene (or which iscomplementary to such an oligonucleotide, or set of oligonucleotides) isidentified (using the above-described procedure), synthesized, andhybridized, by means well known in the art, against a DNA or, a cDNApreparation depending upon the source of the gene. Typically, isolationof eukaryotic genes is done by screening a cDNA library, while a DNAlibrary is used to isolate prokaryotic genes. Techniques of nucleic acidhybridization are disclosed by Maniatis, et al., In: Molecular Cloning,a Laboratory Manual, Second Edition, Coldspring Harbor, N.Y. (1989), andby Haymes, et al., In: Nucleic Acid Hybrization, a Practical Approach,IRL Press, Washington, D.C. (1985), which references are hereinincorporated by reference. The source of DNA or cDNA used willpreferably have been enriched for the desired sequences. Such enrichmentcan most easily be obtained from cDNA obtained by extracting RNA fromcells cultured under conditions which induce erbB-2 ligand synthesis.

Techniques such as, or similar to, those described above havesuccessfully enabled the cloning of genes for human transforming growthfactor-alpha (Derynck, et al., Cell 38:287-298 (1984)), chickenepidermal growth factor receptor (Lax, et al., Mol. Cell. Biol.,8:1970-1978 (1988)), human aldehyde dehydrogenases (Hsu, et al., Proc.Natl. Acad. Sci. USA, 82:3771-3775 (1985)), fibronectin (Suzuki, et al.,Eur. Mol. Biol. Organ. J., 4:2519-2524 (1985)), the human estrogenreceptor gene (Walter, et al., Proc. Natl. Acad. Sci. USA, 82:7889-7893(1985)), tissue-type plasminogen activator (Pennica, et al., Nature,301:214-221 (1983)) and human term placental alkaline phosphatasecomplementary DNA (Kam, et al., Proc. Natl. Acad. Sci. USA, 82:8715-8719(1985)).

In a alternative way of cloning the erbB-2 ligand genes of the presentinvention, a library of expression vectors is prepared by cloning DNA orcDNA, from a cell capable of expressing such a ligand into an expressionvector. The library is then screened for members capable of expressing aprotein which binds to an anti-ligand molecule (antibody or blockingpeptide) and which has a nucleotide sequence that is capable of encodingpolypeptides that have the same amino acid sequence as the erbB-2 ligandprotein of the present invention, or fragments or variants thereof.

Alternatively, DNA sequences encoding erbB-2 ligands may be amplified bythe polymerase chain reaction (PCR) using primers that correspond toappropriate sequences, such as those shown in FIG. 23A. Amplifiedsequences may be introduced into a vector and thereafter cloned asdescribed below.

E. Expression of erbB-2 Ligand Genes

DNA molecules comprising an erbB-2 ligand gene or at least portions ofthis gene can be operably linked into an expression vector andintroduced into a host cell to enable the expression of the ligand bythat cell. Two DNA sequences (such as a promoter region sequence and adesired ligand protein encoding sequence) are said to be operably linkedif the nature of the linkage between the two DNA sequences does not (1)result in the introduction of a frame-shift mutation, (2) interfere withthe ability of the promoter region sequence to direct the transcriptionof the desired protein encoding gene sequence, or (3) interfere with theability of the desired protein gene sequence to be transcribed by thepromoter region sequence.

A DNA sequence encoding an erbB-2 ligand protein may be recombined withvector DNA in accordance with conventional techniques. The presentinvention encompasses the expression of the desired fusion proteins ineither prokaryotic or eukaryotic cells. Eukaryotic hosts include yeast(especially Saccharomyces), fungi (especially Aspergillus), mammaliancells (such as, for example, human or primate cells) either in vivo, orin tissue culture.

Yeast and mammalian cells provide substantial advantages in that theycan also carry out post-translational peptide modifications includingglycosylation. A number of recombinant DNA strategies exist whichutilize strong promoter sequences and high copy number plasmids whichcan be utilized for production of the desired proteins in these hosts.

Yeast recognize leader sequences on cloned mammalian gene products andsecrete peptides bearing leader sequences (i.e., pre-peptides).Mammalian cells provide post-translational modifications to proteinmolecules including correct folding or glycosylation at correct sites.

Mammalian cells which may be useful as hosts include cells of fibroblastorigin such as VERO or CHO-K1, and their derivatives. For a mammalianhost, several possible vector systems are available for the expressionof the desired fusion protein. A wide variety of transcriptional andtranslational regulatory sequences may be employed, depending upon thenature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, etc., may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the genes can be modulated. Ofinterest are regulatory signals which are temperature-sensitive so thatby varying the temperature, expression can be repressed or initiated, orare subject to chemical regulation, e.g., metabolite.

The expression of the desired fusion protein in eukaryotic hostsrequires the use of eukaryotic regulatory regions. Such regions will, ingeneral, include a promoter region sufficient to direct the initiationof RNA synthesis. Preferred eukaryotic promoters include the promoter ofthe mouse metallothionein I gene (Hamer, et al., J. Mol. Appl. Gen.,1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, Cell,31:355-365 (1982)); the SV40 early promoter (Benoist, et al., Nature(London), 290:304-310 (1981)); the yeast gal4 gene promoter (Johnston,et al., Proc. Natl. Acad. Sci. (USA), 79:6971-6975 (1982); Silver, etal., Proc. Natl. Acad. Sci. (USA), 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the desired fusion protein does not containany intervening codons which are capable of encoding a methionine (i.e.,AUG). The presence of such codons results either in the formation of afusion protein (if the AUG codon is in the same reading frame as thedesired fusion protein encoding DNA sequence) or a frame-shift mutation(if the AUG codon is not in the same reading frame as the desired fusionprotein encoding sequence).

The expression of the erbB-2 ligand protein can also be accomplished inprocaryotic cells. Preferred prokaryotic hosts include bacteria such asE. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.Bacterial hosts of particular interest include E. coli K12, and otherenterobacteria (such as Salmonella typhimurium or Serratia marcescens),and various Pseudomonas species. The prokaryotic host must be compatiblewith the replicon and control sequences in the expression plasmid.

To express the desired ligand protein in a prokaryotic cell (such as,for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), itis necessary to operably link the desired ligand protein encodingsequence to a functional prokaryotic promoter. Such promoters may beeither constitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, and the bla promoter of the b-lactamasegene of pBR322, etc. Examples of inducible prokaryotic promoters includethe major right and left promoters of bacteriophage λ (P_(L) and P_(R)),the trp, recA, lacZ, lacI, gal, and tac promoters of E. coli, thea-amylase (Ulmanen, I., et al., J. Bacterial. 162:176-182 (1985)), thes-28 specific promoters of B. subilis (Gilman, M. Z., et al., Gene32:11-20 (1984)), the promoters of the bacteriophages of Bacillus(Gryczan, T. J., In: The Molecular Biology of the Bacilli, AcademicPress, Inc., New York (1982)), and Streptomyces promoters (Ward, et al.,Mol. Gen. Genet., 203:468-478 (1986)). Prokaryotic promoters arereviewed by Glick, B. R., J. Ind. Microbiol., 1:277-282 (1987);Cenatiempo, Y., Biochimie, 68:505-516 (1986); and Gottesman, S., Ann.Rev. Genet., 18:415-442 (1984).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream from the gene-encoding sequence. Suchribosome binding sites are disclosed, for example, by Gold, et al., Ann.Rev. Microbial., 35:365-404 (1981).

The desired protein encoding sequence and an operably linked promotermay be introduced into a recipient prokaryotic or eukaryotic cell eitheras a non-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the desired ligand molecule may occur through the transientexpression of the introduced sequence. Alternatively, permanentexpression may occur through the integration of the introduced sequenceinto the host chromosome.

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay complement an auxotrophy in the host (such as leu2, or ura3, whichare common yeast auxotrophic markers), biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene can either be directly linked to the DNA genesequences to be expressed, or introduced into the same cell byco-transfection.

In a preferred embodiment, the introduced sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to "shuttle" the vector between host cells of different species.

Any of a series of yeast gene expression systems can be utilized.Examples of such expression vectors include the yeast 2-micron circle,the expression plasmids YEP13, YCP and YRP, etc., or their derivatives.Such plasmids are well known in the art (Botstein, et al., Miami Wntr.Symp., 19:265-274 (1982); Broach, J. R., In: The Molecular Biology ofthe Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp. 445-470 (1981); Broach, J. R.,Cell, 28:203-204 (1982)).

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes may be selected byalso introducing one or more markers which allow selection of host cellswhich contain the expression vector. The marker may provide forprototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper or the like. The selectablemarker gene can either be directly linked to the DNA sequences to beexpressed, or introduced into the same cell by co-transformation.Additional elements may also be needed for optimal synthesis of mRNA.These elements may include splice signals, as well as transcriptionpromoters, enhancers, and termination signals. The cDNA expressionvectors incorporating such elements include those described by Okayama,H., Mol. Cell. Biol., 3:280 (1983), and others.

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli such as, for example, pBR322, ColE1, pSC101,paCYC 184, πVX. Such plasmids are, for example, disclosed by Maniatis,et al., (In: Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1982)). Bacillus plasmids includepC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T.(In: The Molecular Biology of the Bacilli, Academic Press, New York(1982), pp. 307-329). Suitable Streptomyces plasmids include pIJ101(Kendall, et al., J. Bacterial., 169:4177-4183 (1987)), and Streptomycesbacteriophages such as φC31 (Chater, et al., In: Sixth InternationalSymposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary(1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, et al.,(Rev. Infect. Dis., 8:693-704 (1986)), and Izaki, K. (Jpn. J.Bacteriol., 33:729-742 (1978)) .

Once the vector or DNA sequence containing the construct has beenprepared for expression, the DNA construct may be introduced(transformed) into an appropriate host. Various techniques may beemployed, such as protoplast fusion, calcium phosphate precipitation,electroporation or other conventional techniques. After the fusion, thecells are grown in media and screened for appropriate activities.Expression of the sequence results in the production of the recombinanterbB-2 ligand protein of the present invention.

F. Purification of Recombinant erbB-2 Ligand

The erbB-2 ligand proteins of this invention can be produced byfermentation of the recombinant host containing the cloned ligand genes.The recombinant host, such as mammalian cells producing the clonedprotein, can be grown and harvested according to techniques well knownin the art.

The recombinant erbB-2 ligand proteins of the present invention can beextracted and purified from the recombinant host or its culture media byusing known protein purification techniques commonly employed, such asextraction, precipitation, ion exchange chromatography, affinitychromatography, gel filtration and the like. Biochemical techniquesemployed to isolate the erbB-2 ligand proteins of the present inventionfrom SK-Br-3 are of particular interest when purifying these proteinsfrom a recombinant host.

G. Anti-Ligand Molecules

The present invention concerns anti-ligand molecules which bindcovalently or non-covalently to the erbB-2 ligands of the presentinvention. Without being limited, the anti-ligand molecules of thepresent invention include antibodies, blocking peptides and any othermolecule, compound, chemical, etc. that is capable of covalently ornon-covalently binding to the erbB-2 ligand of the present invention.

Blocking peptides, according to the present invention, are "capable ofbinding" a molecule if they are capable of specifically reacting with orhave affinity for the molecule such that the blocking peptide will bindto the molecule. An example of a blocking peptide of the presentinvention is the extracellular domain of the erbB-2 transmembranereceptor. Typically, peptide fragments of the extracellular domain whichbind to the erbB-2 ligand of the present invention may be used, althoughfunctional derivatives of such erbB-2 transmembrane receptor may beused. Such derivatives may include, for example, neu, c-kit, met or anytransmembrane tyrosine kinases that have a structure reminiscent ofgrowth factor receptors.

The blocking peptides of the present invention may be prepared by anumber of well known techniques. Synthetic peptides may be constructedusing automated protein synthesizers. Alternatively, the blockingpeptides of the invention may be generated through recombinant DNAtechniques. For instance, a DNA molecule encoding for the desiredpeptide may be operably linked to a promoter and other regulatorysequences such that expression of said peptide can be obtained in atransformed host. A number of methods of producing a desired blockingpeptide will be readily apparent to one of skill in the art.

It will be understood by those of skill in the art that the blockingpeptide of the present invention can be detectably labeled or conjugatedwith therapeutic agents by standard techniques well known in the art.Examples of detectable labels are described below which may be used todetectably label the blocking peptides of the present invention.

The term "therapeutic agent" as used herein is meant to refer to anymolecule, chemical compound, protein etc. which, when introduced inclose association to a cell, is capable of killing, destroying,inhibiting the growth or reproduction of, or otherwise interfering inthe normal physiology or metabolism of said cell in a manner notconducive to the cell's survival or reproduction. Examples of suitabletherapeutic agents include cytotoxic drugs, toxins, isotopes, endocrinetherapies and the like. Specific cytotoxic drugs that may be used areAdriamycyn, Cyclophosphamide, 5-Fluorouracil, Methotrexate, Cisplatin,Carboplatin, Vincristine, VP-16, Bleomycin, Mitomycin C, Taxol, etc.Toxins may include Ricin A, Diphtheria, and Pseudomonas. Examples ofsuitable isotopes include p³², Indium, Yttrium, and Iodine. Examples ofsuitable endocrine therapy include Diethyl bestrol (DES), Tamoxifen, andLHRH antagonizing drugs.

The term "antibody" encompasses whole immunoglobulin as well asimmunoglobulin fragments. "Antibody" (Ab) or "polyclonal antibody"and/or "monoclonal antibody" (Mab) as used herein is meant to includeintact molecules, such as immunoglobulin G molecules made up of fourimmunogobulin peptide chains, two heavy chains and two light chains, aswell as fragments thereof (such as, for example, Fab and F(ab')2fragments) which are capable of binding a hapten or antigen."Immunoglobulin fragments" are protein molecules related toimmunoglobulin, which are known to retain the epitopic bindingspecificity of the original antibody, such as Fab, F(ab)'₂, Fv, etc. Faband F(ab')₂ fragments lack the Fc fragment of intact antibody, clearmore rapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody (Wahl, et al., J. Nucl. Med., 42:316-325(1983)).

An antibody is said to be "capable of binding" or "directed against" amolecule if it is capable of specifically reacting with the molecule tothereby bind the molecule to the antibody. The term "epitope" or"binding site" is meant to refer to that portion of an antigen which canbe recognized and bound by an antibody. An antigen may have one, or morethan one epitope. An "antigen" is capable of inducing an animal toproduce antibody capable of binding to an epitope of that antigen. Thespecific reaction referred to above is meant to indicate that theantigen will react, in a highly selective manner, with its correspondingantibody and not with the multitude of other antibodies which may beevoked by other antigens. The antigen of the present invention can beany erbB-2 ligand identified herein, including erbB-2 ligand fragmentsand synthetic peptides which have amino acid sequences corresponding toerbB-2 ligand sequences. For example, the p75 erbB-2 ligand can be usedto generate anti-p75 ligand antibodies according to the presentinvention.

The antibodies used in the present invention may be prepared by any of avariety of methods. For example, cells producing erbB-2 ligand (orfractions, lysates, etc. thereof) can be administered to an animal inorder to induce the production of sera containing polyclonal antibodiesthat are capable of binding the antigen. Since cells which produceerbB-2 ligand excrete the protein into the culture media, the media maybe used as a source of the erbB-2 ligand antigen. In a preferred method,a preparation of the erbB-2 ligand of the present invention is preparedand purified to render it substantially free of natural contaminants.Particularly preferred preparations of erbB-2 ligand are produced byexpression of recombinant DNA encoding an erbB-2 ligand in a non-humanhost cell, since antigens that are found with the erbB-2 ligand in thenative state will not be expressed by the recombinant host. Suchpreparations are then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

The antibodies of the present invention may be monoclonal or polyclonalantibodies (or hapten binding fragments thereof). Such monoclonalantibodies can be prepared using hybridoma technology (Kohler, et al.,Nature, 256:495 (1975); Kohler, et al., Eur. J. Immunol., 6:511 (1976);Kohler, et al., Eur. J. Immunol., 6:292 (1976); Hammerling, et al., In:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981)). In general, such procedures involve immunizing an animal withsubstantially pure erbB-2 ligand protein.

The splenocytes of the immunized animal are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, a suitableparent myeloma cell line (SP₂ O), available from the American TypeCulture Collection, Rockville, Md., may be used. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium, andthen cloned by limiting dilution as described by Wands, et al.,Gastroenterology, 80:225-232 (1981), which reference is hereinincorporated by reference). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding to the erbB-2 ligand.

It will be appreciated that Fab and F(ab')₂ and other fragments of theantibody may be used according to the methods disclosed herein for thedetection erbB-2 ligand in samples in the same manner as intactantibody. Such fragments are typically produced by proteolytic cleavage,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab')₂ fragments). Alternatively, hapten-binding fragments canbe produced through the application of recombinant DNA technology orthrough synthetic chemistry.

Similar to blocking peptides, antibodies can be conjugated to thetherapeutic agents. Suitable examples of therapeutic agents which may beconjugated to the anti-ligand antibodies of the present inventioninclude, but are not limited to, cytotoxic drugs, toxins, and isotopes.Examples of suitable therapeutic agents are described above.

The polyclonal or monoclonal antibodies produced against gp30 or p75 maybe produced in accordance with well-known techniques. For example, seeCurrent Protocols in Molecular Biology, edited by F. M. Ausubel, et al.(Wiley 1987), in particular Chapter 11 on Immunology. Also, theimmunoassays used in the assays and diagnostic test kits of the presentinvention are well known to the artisan as evidenced by the abovetreatise, and by the methods disclosed in U.S. Pat. No. 4,921,790 whichpatent has been specifically incorporated herein in the entirety.

H. Assays for Detecting erbB-2 Ligand

The 185 kd transmembrane glycoprotein known as p185^(erbB-2) is thoughtto be a transmembrane protein which functions as a growth factorreceptor and is encoded by a protooncogene. The erbB-2 expression isamplified in many adenocarcinomas and, in particular, is amplified oroverexpressed in nearly 30% of human breast cancers (Maggurie, et al.,Seminars in Oncology, 16:148-155 (1989)). Patients with cancer cellswhich overexpress erbB-2 are known to have much shorter disease-freeperiods and poorer overall survival than cancer patients that do notshow erbB-2 overexpression. Consequently, it is important to distinguishbetween malignancies which exhibit erbB-2 overexpression from thosewhich do not. Diagnosis of erbB-2 associated cancers thus provide theclinician with a way to pre-select an effective therapy for treatingparticular types of cancer.

Expression of the erbB-2 protooncogene encoding a 185 kDa transmembraneprotein serves as a marker to identify a particular invasive malignantcell type. Since the erbB-2 receptor is a transmembrane protein, itsextracellular domain is accessible to interaction with its ligand on thecell surface. Consequently, the ligand of the present invention whichbinds specifically to p185^(erbB-2) can be utilized to detect cellswhich express the erbB-2 receptor. Surprisingly, the erbB-2 ligand ofthe present invention does not cross-react with the EGFR and thus isspecific for erbB-2 receptor. Therefore, the erbB-2 ligands of theinvention are capable of detecting particular cancer cells in a patientand may not recognize normal cells or malignant cells that fail tooverexpress the erbB-2 receptor. This characteristic is important inthat early detection of erbB-2 overexpressing malignant cells mayindicate prognosis and treatment for the patient.

According to the present invention, diagnosis with the erbB-2 ligandsinvolves the detection of p185^(erbB-2) overexpressing cancer cells in apatient. Detection of such cells in a patient may be accomplished by anyof a variety of in vitro assays or in vivo imaging techniques. Examplesof these in vitro and in vivo techniques are disclosed in the preferredembodiments described below. The materials for use in the in vitroassays and in vivo imaging techniques which utilize erbB-2 ligand arealso ideally suited for preparation of a kit.

The anti-ligand molecules including antibodies, fragments of antibodies,or blocking peptides of the present invention may be used to detect thepresence of the erbB-2 ligand. Thus, the antibodies (or fragmentsthereof) and blocking peptides may be employed in histology and biopsyto detect erbB-2 ligand expression in a patient suffering from breast,liver, ovarian, lung, colon carcinomas and the like. Such detection maybe accomplished using any of a variety of assays. For example, byradioactively labeling the antibodies or antibody fragments, it ispossible to detect the erbB-2 ligand through the use of radioimmuneassays. A good description of a radioimmune assay (RIA) may be found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work, etal., North Holland Publishing Company, New York (1978), with particularreference to the chapter entitled "An Introduction to Radioimmune Assayand Related Techniques" by Chard, T., incorporated by reference herein.Alternatively, fluorescent, enzyme, or other suitable labels can beemployed. Detectably labeled blocking peptides may be used in ananalogous manner to detect the erbB-2 ligand.

The present invention also provides a method of detecting cells whichoverexpress p185^(erbB-2) in a patient, which generally entailscontacting a sample obtained from the patient with a detectably labellederbB-2 ligand; and detecting the presence of the erbB-2 ligand in thesample using some minor modification of standard radioimmunoassay orradio receptor assay methodology. Generally, the sample may be any oneof or combination of the following: body tissue, body fluids such asblood, urine, saliva, tear drops, serum, and cerebrospinal fluid, and/orfeces.

Alternatively, the detection of erbB-2 ligand may be accomplished by invivo imaging techniques, in which the labeled antibodies, fragmentsthereof, or blocking peptides are provided to a patient, and thepresence of the breast, ovarian, liver, lung, or colon carcinoma whichexpresses erbB-2 ligand is detected without the prior removal of anytissue sample. Such in vivo detection procedures have the advantage ofbeing less invasive than other detection methods, and are, moreover,capable of detecting the presence of antigen-expressing cells in tissuewhich cannot be easily removed from the patient.

In accordance with the above-discussed assays, antibodies, fragmentsthereof, or blocking peptides may be labeled using any of a variety oflabels and methods of labeling. Examples of types of labels which can beused in the present invention include, but are not limited to, enzymelabels, radioisotopic labels, non-radioactive isotopic labels,fluorescent labels, toxin labels, and chemiluminescent labels.

Examples of suitable enzyme labels include malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcoholdehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphateisomerase, peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase,etc.

Examples of suitable radioisotopic labels will be readily apparent toone of skill in the art. Suitable non-radioactive isotopic labels foruse in the present invention will also be known to one of ordinary skillin the art.

Examples of suitable fluorescent labels include a fluorescein label, anisothiocyanate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an o-phthaldehyde label, afluorescamine label, etc.

Examples of suitable toxin labels include diphtheria toxin, ricin,pseudomonas endotoxin and cholera toxin. Examples of chemiluminescentlabels include a luminal label, an isoluminal label, an aromaticacridinium ester label, an imidazole label, an acridiniu salt label, anoxalate ester label, a luciferin label, a luciferase label, an aequorinlabel, etc.

Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedy,J. H., et al. (Clin. Chim. Acta, 70:1-31 (1976)), and Schurs, et al.(Clin. Chim. Acta, 81:1-40 (1977)). Coupling techniques mentioned in thelatter are the glutaraldehyde method, the periodate method, thedimaleimide method, the m-maleimido-benzyl-N-hydroxy-succinimide estermethod, all of which methods are incorporated by reference herein.

The detection of the antibodies, fragments of antibodies or blockingpeptides can be improved through the use of carriers. Well-knowncarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen. Thus, the support configuration may be spherical,as in a bead, or cylindrical, as in the inside surface of a test tube,or the external surface of a rod. Alternatively, the surface may be flatsuch as a sheet, test strip, etc. Those skilled in the art will notemany other suitable carriers for binding antibodies and blockingpeptides, or will be able to ascertain the same by use of routineexperimentation.

The binding molecules (anti-ligand molecules) of the present inventionmay also be adapted for utilization in an immunometric assay, also knownas a "two-site" or "sandwich" assay. In a typical immunometric assay, aquantity of unlabeled antibody, or fragment of antibody, is bound to asolid support that is insoluble in the fluid being tested (i.e., blood,lymph, liquified, stools, tissue homogenate, etc.) and a quantity ofdetectably labeled soluble antibody is added to permit detection and/orquantitation of the ternary complex formed between solid-phase antibody,peptide antigen, and labeled antibody. It will be apparent to one ofskill that labeled blocking peptide may also be used in place of orcombination with the antibody used in the assay according to theinvention.

Typical immunometric assays include "forward" assays in which theantibody or blocking peptide bound to the solid phase is first contactedwith the sample being tested to extract the antigen from the sample byformation of a binary solid phase antibody-antigen complex or a blockingpeptide-antigen complex. After a suitable incubation period, the solidsupport is washed to remove the residue of the fluid sample, includingunreacted antigen, if any, and then contacted with the solutioncontaining an unknown quantity of labeled antibody or labeled blockingpeptide (which functions as a "reporter molecule"). After a secondincubation period to permit the labeled molecule to complex with theantigen bound to the solid support through the unlabeled antibody orblocking peptide, the solid support is washed a second time to removethe unreacted labeled antibody. This type of forward sandwich assay maybe a simple "yes/no" assay to determine whether erbB-2 ligand antigen ispresent or may be made quantitative by comparing the measure of labeledantibody or labeled blocking peptide with that obtained for a standardsample containing known quantities of antigen. Such "two-site" or"sandwich" assays are described by Wide at pages 199-206 of RadioimmuneAssay Method, edited by Kirkham and Hunter, E. & S. Livingstone,Edinburgh, 1970.

In another type of "sandwich" assay, which may also be useful to detectthe erbB-2 ligand antigen, the so-called "simultaneous" and "reverse"assays are used. A simultaneous assay involves a single incubation stepas the antibody or blocking peptide bound to the solid support andlabeled antibody or blocking peptide are both added to the sample beingtested at the same time. After the incubation is completed, the solidsupport is washed to remove the residue of fluid sample and uncomplexedlabeled antibody or peptide. The presence of labeled antibody orblocking peptide associated with the solid support is then determined asit would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition of a solution of labeledantibody or labeled blocking peptide to the fluid sample is followed bythe addition of unlabeled antibody or unlabeled blocking peptide boundto a solid support after a suitable incubation period is utilized. Aftera second incubation, the solid phase is washed in conventional fashionto free it of the residue of the sample being tested and the solution ofunreacted labeled antibody or block peptide. The determination oflabeled antibody or labeled blocking peptide associated with a solidsupport is then determined as in the "simultaneous" and "forward"assays.

As explained above, the immunometric assays for erbB-2 ligand antigenrequire that the particular binding molecule be labeled with a "reportermolecule." These reporter molecules or labels, as identified above, areconventional and well-known to the art. In the practice of the presentinvention, enzyme labels are a preferred embodiment. No single enzyme isideal for use as a label in every conceivable immunometric assay.Instead, one must determine which enzyme is suitable for a particularassay system. Criteria important for the choice of enzymes are turnovernumber of the pure enzyme (the number of substrate molecules convertedto the product per enzyme site per unit of time), purity of the enzymepreparation, sensitivity of detection of its product, ease and speed ofdetection of the enzyme reaction, absence of interfering factors or ofenzyme-like activity in the test fluid, stability of the enzyme and itsconjugate, availability and cost of the enzyme and its conjugate, andthe like. Included among the enzymes used as preferred labels in theimmunometric assays of the present invention are peroxidase, alkalinephosphatase, beta-galactosidase, urease, glucose oxidase, glycoamylase,malate dehydrogenase, and glucose-6-phosphate dehydrogenase.

In addition, the materials for use in the assays of the invention areideally suited for preparation of a kit. Such a kit may comprise acarrier means being compartmentalized to receive in close confinementone or more container means such as vials, test tubes, and the like.Each of said container means comprises one of the separate elements tobe used in the method.

For example, one of said container means may comprise animmuno-absorbent-bound peptide fragment. Such fragment may be bound to aseparate solid-phase immuno-absorbent or directly to the inner walls ofa container. A second container may comprise detectably labeled antibodyor blocking peptide in lyophilized form or in solution.

The carrier may also contain, in addition, a plurality of containerseach of which comprises different, predetermined and known amounts ofantigen. These latter containers can then be used to prepare a standardcurve from which can be interpolated the results obtained from thesample containing the unknown amount of antigen.

One of skill in the art will recognize that in accordance with theseassays, a variety of labels and methods of labeling may be used.Examples of types of labels that can be used in the present inventioninclude, but are not limited to, enzyme labels, radioisotopic labels,non-radioactive isotopic labels, fluorescent labels, toxin labels, andchemiluminescent labels. The binding of these labels to the erbB-2ligand protein may be accomplished using standard techniques commonlyknown to those of ordinary skill in the art.

As gp30 is known to be produced by MDA-MB-231 breast cancer cells, andis also likely to be produced by other adenocarcinoma cancer cells, thepresent invention also provides a method for detecting gp30 in patientsera or urine or other body fluids.

Generally, the present conjugates may be used advantageously in abiochemical detection method in which the 30 kDa glycoprotein ligand isbound to a surface and put into contact with aqueous solution containinga tumor portion containing cells which are suspected of overexpressingeither EGFR or erbB-2 oncogene. This is conveniently done as either EGFRor p185 may be found on the cell surfaces. If such cells are present,either the EGFR or p185^(erbB-2) will become bound to the ligand.Thereafter, the aqueous solution is separated from the bound antiligandmaterial, and the antiligand material may be conveniently detected witha known detection means associated therewith. For example, an amplifiedenzyme-linked immunoassay may be used. The surface to which the ligandis bound is treated with one or more agents for limiting the amount ofnon-specific binding. Such agents reduce the "noise" arising due tonon-specific binding when interpreting the assay.

In accordance with the above procedure, a diagnostic test kit may beconstructed in a variety of ways. For example, a test kit may beconstructed to contain a vessel containing a test liquid having asurface to which gp30 ligand is bound. This is preferably a multi-welltest plate. Also contained is at least one other vessel containingreagent solution. The agent for limiting non-specific binding may beincorporated within a solution of the kit or may have been used to treatthe surface of the first vessel before it is supplied. Then, a portionof the tumor or a tumor sample may be worked up into an aqueous solutionand put into contact with the bound gp30.

In order to conveniently detect the overexpression of EGFR or erbB-2oncogene in a human patient it is advantageous to use the well-knownsandwich assay technique. For example, one assay method and test kitwhich may be used in accordance with the present invention are describedin U.S. Pat. No. 4,668,639 which is incorporated herein in the entirety.

Hence, the present invention contemplates and is specifically directedto any diagnostic or therapeutic method for the detection ofadenocarcinoma cells which overexpress EGFR or erbB-2 oncogene, whichmethod uses the formation of a conjugate between the 30 kDa glycoproteinof the present invention and either EGFR or p185^(erbB-2).

As noted above, the present invention also provides an assay and a testkit for the detection of gp30 using polyclonal or monoclonal antibodiesto gp30. Importantly, however, the presence of the 30 kDa glycoprotein(gp30) in patient sera can be detected utilizing either monoclonal orpolyclonal antibodies in virtually any type of immunoassay. Thisincludes both single-site or two-site or "sandwich" assays of thenon-competitive types, as well as in traditional competitive bindingassays. In such an assay, the monoclonal antibodies to gp30arepreferably bound to the microtiter or multi-well plate and exposed topatient sera suspected of containing gp30. Upon detecting the presenceof gp30 by a conventional detecting means, a conclusion of poorprognosis would be made necessitating the use of more aggressivetreatment for the tumor.

With the above assay, a test kit is also provided. Generally, the kitcontains a first container containing an antibody having specificity forgp30 and a second container containing a second antibody havingspecificity for gp30 and being labelled with a reporter molecule capablegiving a detectable signal. The first antibody is immobilized on a solidsurface. The above assay and test kit for the detection of gp30 may be,respectively, conducted and constructed by analogy in accordance withU.S. Pat. No 4,921,790, which is incorporated herein in the entirety.

As described above, the diagnostic aspects of the present inventionrelate to the use of methods and test kits for the detection of eitherp185^(erbB-2), EGFR or gp30. The detection of any one of these proteinsmay form the basis for a poor prognosis necessitating the use ofaggressive treatment of one or more adenocarcinomas.

Diagnostic uses of the anti-ligand molecules of the present inventionmay include, for example, detection of erbB-2 ligand in a sampleobtained from a patient. Such samples may be body tissue, body fluids(such as blood, urine, tear drops, saliva, serum, and cerebrospinalfluid), feces, cellular extracts and the like. According to the methodof detecting erbB-2 ligands, the erbB-2 ligand of the present inventionis excreted in vitro into cell culture medium. Another growth factor,TGFα, also secreted in vitro was identified in body fluids of cancerpatients. Consequently, the growth factor of the present invention(erbB-2 ligand) may be detected in body fluids, stools, etc. from acancer patient.

Assaying for the erbB-2 ligand of the invention in a sample obtainedfrom a patient may thus provide for a method for diagnosing cancer. Thatis, detection of erbB-2 ligand in a sample obtained from a patientindicates the presence of erbB-2 ligand-expressing cells in a patient.Cancer patients with adenocarcinoma cells that overexpress the erbB-2receptor are known to have a much shorter disease-free period and pooreroverall survival than cancer patients that do not show erbB-2overexpression. Detection of erbB-2 ligand growth factor may thus serveas a prognostic test, allowing the clinician to select a more effectivetherapy for treating the patient.

I. Therapeutic Uses of erbB-2 Ligand

The erbB-2 ligand of the present invention may be used bothdiagnostically and therapeutically. Specifically, the erbB-2 ligand maybe used to detect, in a patient, adenocarcinoma cells which overexpressthe erbB-2 receptor protein. Treatment of such a patient to growthinhibit or destroy these cells may also be accomplished according to thepresent invention.

The erbB-2 ligand of the present invention may be used to treat apatient suffering from cancer. Treatment therapies with erbB-2 ligandare specifically targeted against cells which may bind to the erbB-2ligand of the present invention. In this manner, malignant cells thatoverexpress the erbB-2 receptor (or related receptors) may be growthinhibited or destroyed by the treatment method of the present invention.It will be appreciated that a number of therapeutic uses of the erbB-2ligand of this invention may be devised. Thus, the present invention isnot meant to be limited to the therapeutic treatments described, andthey are only presented by way of illustration.

One aspect of cancer treatment using the erbB-2 ligand of this inventionconcerns the use of ligand-therapeutic agent conjugates. The erbB-2ligand conjugates of the invention may bind to the adenocarcinoma cellwhich overexpress the erbB-2 receptor. Once the erbB-2 ligand conjugateis bound to the cell, the therapeutic agent is capable of killing orinhibiting the growth of that cell.

In this manner, administration of an effective amount of ligandconjugate to a patient serves as a treatment that may destroy or inhibitgrowth of particular types of cancer cells in vivo. Normal cells are notaffected by administration of the ligand-therapeutic agent conjugate ofthis invention.

A second aspect of treatment using the erbB-2 ligand of the presentinvention relates to the inhibitory affects of the growth factor.Surprisingly, the erbB-2 ligand of the present invention acts, insufficient concentrations, as an inhibitor capable of inhibiting orsuppressing proliferation of adenocarcinoma cells. Any of a number ofcancer cells may be growth inhibited with the erbB-2 ligand of thepresent invention, provide that the erbB-2 ligand can interact with thecell. Typically, malignant cells which overexpress the erbB-2 receptorare inhibited. Such cells may include, but are not limited to, breast,lung, ovarian, gastric, thyroid, prostate or salivary gland carcinomacells. Cells not affected by the erbB-2 ligand of the invention includenormal cells and malignant cells which do not overexpress theprotooncogene coding for the erbB-2 receptor.

In vitro or in vivo inhibition of tumor cells may be accomplished byadministration of an effective amount of the erbB-2 ligand of thepresent invention. One of skill in the art will recognize that theamount sufficient to inhibit cell growth varies depending on the celltype, the body weight of the patient, the type of therapeutic agent usedetc. These variables can easily be determined by those skilled in theart with little experimentation.

For example, the 30 kDa glycoprotein of the present invention may beused advantageously to inhibit the growth of various types ofadenocarcinoma cells which overexpress the erbB-2 oncogene and/or EGFR.Preferably, the present 30 kDa glycoprotein is used in inhibit thegrowth of adenocarcinoma cells of breast, ovarian, gastric and lungtissue which overexpress the erbB-2 oncogene and EGFR.

The therapeutic aspects of the present invention relate to the use ofgp30 to inhibit the growth of adenocarcinoma cells which overexpressEGFR and/or erbB-2 oncogene. Generally, the amount of gp30 to beadministered as a therapeutic agent will be determined on a case by casebasis by the attending physician. As a guideline, the extent of theadenocarcinoma, body weight and age of the patient are considered whileup to about 10,000 ng per day may be used, generally not more than 1,000ng per day of gp30 is administered. It is preferred, however, if fromabout 5-500 ng per day are used. Notably, however, the above amounts mayvary on a case-by-case basis.

In using the present 30 kDa glycoprotein to control the growth of theabove malignant cells in a mammal, preferably a human, relatively highor low concentrations of the glycoprotein may be used. For example, tostimulate cell growth, an aqueous solution having a concentration ofabout 1-50 ng/ml may be conveniently administered to a patient such thata total of from about 1-10,000 ng of glycoprotein are administered perday. It is preferred, however, if about 1-1,000 ng are administered perday. Alternatively, to inhibit cell growth, an aqueous solution having aconcentration greater than 1 mg/ml may conveniently be administered to apatient such that concentrations are achieved which will directlyinhibit overexpressing cells.

While the present 30 kDa glycoprotein may be administered by itself, asa therapeutic agent, it may be administered in combination with one ormore other therapeutic agents. For example, the 30 kDa glycoprotein maybe administered with any chemotherapeutic substance, growth inhibitor orimmune-stimulating substance. The present invention specificallycontemplates such combinations. gp30, when used in very low (pm)concentrations, may be used to simulate the growth of cellsoverexpressing erbB-2.

As indicated above, however, the amount of gp30 to be administered foran inhibitory or stimulatory effect will generally be determined on acase-by-case basis by the attending physician. Generally, a lesseramount of gp30 is required to exhibit a stimulatory effect than isrequired to exhibit an inhibitory effect. The appropriate amount may bereadily determined using human cell samples or by considering variousfactors for the patient on a case-by-case basis.

Further, the gp30 of the present invention is generally administered insolution form by intravenous injection using a solution having aconcentration of gp30 of about 0.1 mg to 1 mg per ml of solution. Thesolution may be an aqueous solution, a saline solution as used inclinical practice or dextrose 5% saline solution.

Thus, the present invention provides a substantially pure erbB-2 ligandor functional derivative thereof, which ligand has a molecular weight ofabout 30 Kd, and which is capable of binding to erbB-2 receptor protein,p185^(erbB-2).

Generally, in accordance with the present invention, at the lower end ofthe concentrations expressed above, such as in the range of about 0.05ng to about 1 μg, preferably about 0.1 ng to about 1 ng, the presenterbB-2 ligand conjugate stimulates the growth of malignant cells whichexpress erbB-2.

The use of erbB-2 ligands that do not bind to EGFR such as p75 may bepreferred for treatment of some cancers. The above procedures fortherapy will also apply to such ligands.

The amounts of erbB-2 ligand of the present invention used in treatmentof cancer will generally be related to the amount of erbB-2 ligand thathas been shown to be effective when administered to cells of the sametype in vitro. Levels of erbB-2 ligand that induce proliferation ofcells in vitro are considered stimulatory, while concentrations thatinduce differentiation of cells in vitro are generally consideredinhibitory. The erbB-2 ligand will be administered in a manner andamount so that the circuating concentration of the ligand is similar tothe concentration found to be effective in vitro. Generally, thecirculating concentration will be from about 0.05 ng/ml to 1 μg/ml,where stimulatory concentrations are generally below 1 ng/ml andinhibitory concentrations are generally above about 1 ng/ml.

J. Therapeutic Uses of Anti-Ligand Molecules

The anti-ligand molecules of the present invention have a multitude oftherapeutic and diagnostic uses. For example, therapeutic uses mayinvolve cancer therapy in a patient suspected of suffering from cancer.Specifically, the anti-ligand molecules of the present invention such asantibodies or blocking peptides may be used to treat patients that haveadenocarcinoma cells which produce the erbB-2 ligand and/or overexpressthe erbB-2 receptor proteins.

One type of treatment may involve the use of the antibody conjugated toa therapeutic agent. Blocking peptide coupled to a therapeutic agent maybe used in an analogous manner. By administering an effective amount ofanti-ligand coupled to the therapeutic agent to a patient, theadenocarcinoma cells in the patient which express erbB-2 ligand and/or aerbB-2 receptor can be growth inhibited or killed, thereby providing atreatment for cancer. Normal and malignant cells which overexpress EGFRare not affected by administration of the anti-ligand-therapeuticconjugate agent to a patient. Thus, treatment of a patient with theanti-ligand conjugate may selectively inhibit or destroy erbB-2overexpressing cancer cells in vivo.

In accordance with the method of cancer treatment of the invention, theconjugated anti-ligand is capable of recognizing and binding tocarcinoma cells due to the carcinoma cells association with the erbB-2ligand. Without being limited, the mechanism of binding to the cancercell may involve the recognition of erbB-2 ligand located on the cellsurface or because of expression and/or secretion of the ligand.

Once the conjugated anti-ligand is bound or in close association withthe adenocarcinoma cell by interacting with ligand, the therapeuticagent is capable of inhibiting or killing that cell. In this manner, thetherapy of the present invention is selective for a particular target,i.e., cancer cells which are associated with the erbB-2 ligand. Normalcells and other cells not associated with the erbB-2 ligand (cells whichdo not express or bind erbB-2 ligand) may not, for the most part, beaffected by this therapy.

Alternatively, the anti-ligands of the present invention may be used toprevent or inhibit inducement of adenocarcinoma cell proliferation. Forexample, cancer cells which contain the p185^(erB-2) receptor areinduced to proliferate in the presence of low concentrations of erbB-2ligand. Preventing the erbB-2 ligand growth factor from interacting withits receptor may provide a means to treat a cancer patient.

According to the method of inhibiting cellular proliferation of thepresent invention, the anti-ligand is capable of binding to the erbB-2ligand. Binding the excreted erbB-2 ligand in vivo forms aligand-anti-ligand complex and thus may prevent or inhibit theligand-receptor interaction either sterically or otherwise. Thus, thepresent invention provides a treatment to prevent or inhibitadenocarcinoma cell proliferation in a patient by administering aneffective amount of an anti-ligand to such a patient.

It will be appreciated that a number of other therapeutic uses of theanti-ligands of the present invention may be devised. Such therapies mayinvolve use of other known treatment techniques in combination with theanti-ligands of the invention. The present invention is not meant to belimited by the therapeutic treatments described which are only presentedby way of illustration.

Furthermore, administration of an effective amount of the anti-ligandsof the present invention sufficient to inhibit or kill an adenocarcinomacell may vary depending upon a number of factors including the type ofmalignant cell, body weight of the patient, the type of therapeuticagent used and the like. Those of skill in the art will appreciate thatthe amount necessary to inhibit or kill a particular malignant cell invitro or in vivo can easily be determined with minimal experimentation.

In order to further exemplify the present invention, reference will nowbe made to certain examples which are provided solely for purposes ofillustration and are not intended to be limitative.

Cell Lines

Cells from the following sources were used in the Examples: MDA-MB-231SKBr-3, MDA-MB-453, BT-474, and NRK clone 49F fibroblasts were obtainedfrom the American type Culture Collection (Rockville, Md.). Hs578Tcells, A431 cells, and H8 cells, a TGFα-transfected MCF-7 breast cancercell line, were available upon request from a variety of sources.Carcinogen-immortalized normal mammary epithelial cell subline 184A1N4and its SV40-transfected derivative 184A1N4T, were also available onrequest. Rat-FeSrV transfected cells were also provided upon request.All cell lines were propagated in improved modified Eagle's medium(IMEM, Gibco, Grand Island N.Y.) supplemented with 10% fetal bovineserum (FBS, Gibco).

Conditioned Media Preparation, Collection and Concentration

Conditioned media collections were carried using a well-known procedure.The media were concentrated 100-fold in an Amicon ultra-filtration cell(YM5 membrane) (Amicon, Denvers, Mass.). Once clarified andconcentrated, the media were stored at -20° C. while consecutivecollections were made during the following days. The concentrated mediawere dialyzed using Spectraphore 3 tubing (Spectral Medical Industries,Los Angeles, Calif.) against 100 volumes of 0.1M acetic acid over atwo-day period at 4° C. The material that precipitated during dialysiswas removed by centrifugation at 4000 rpm for 30 min. at 4° C.; proteaseinhibitors were added. The clarified sample was then lyophilized. Thefollowing Examples are provided solely for the purpose of illustratingthe present invention and are not intended to be limitative.

In order to further describe the various aspects of the presentinvention, reference will now be made to the figures of the presentspecification.

EXAMPLE 1 Identification of a TGFα-like Polypeptide in MDA-MB-231 HumanBreast Cancer Cells

To determine whether the 30 kDa TGFα-like protein was recognized byantibodies developed against mature 6 kDa TFGα, MDA-MB-231 cells weremetabolically labelled with ³⁵ S! methionine and ³⁵ S! cysteine.

Cells were grown to 80% confluence in IMEM. Cell monolayers were washedthree times with PBS and incubated for two hours in serum-free IMEMwhich lacked methionine and cysteine and was supplemented with glutamine(2.9 g/1) (Biofluids, Rockville, Md.). This medium was then removed andreplaced with serum-free IMEM without methionine and cysteine containing2.5 mCi/ml ³⁵ S! cysteine and methionine (Amersham, Arlington Heights,Ill., 1175 Ci/mmole). A total of 2.5 ml of this medium was used for a 5cm dish. The medium was harvested from the culture after 16 hrs at 37°C. and clarified by centrifugation. Cells were washed once with PBS,harvested by scraping, and lysed in 1 ml of RIPA buffer (300 mM NaCl,100 mM Tris-HCl, containing 2% Triton X 100, 2% Nadeoxycholate, 0.2%SDS, 0.4% BSA and 2 mM PMSF). Following an incubation of 30 minutes onice, the lysate was clarified by centrifugation (30 minutes at 4000 rpm)and used immediately or was stored at -70° C.

Metabolically labelled conditioned media from MDA-MB-231,TGFα-transfected MCF-7 (H8), and HS578T cells were tested by solid phaseRIA for immunoreactivity with a polyclonal antibody (R399) and amonoclonal antibody raised against recombinant 6 kDa TGFα.

Polyclonal and Monoclonal Antibodies

Polyclonal Antibodies Antiserum against human TGFα was obtained byimmunization of a rabbit on day 0 with 400 g of recombinant TGFαsynthesized in E. coli, provided by Genentech Corp. The immunogen wasfirst conjugated to keyhole limpet hemocyanin (KLH) and was emulsifiedin complete Freund's adjuvant and was injected intradermally at multiplesites. Additional injections were given as follows: day 60, 175 g TGFαand days 90, 150, 180, and 210, 100 g TGFα. The booster injections weregiven subcutaneously at multiple sites in incomplete Freund's adjuvant.The rabbit serum was assayed for antibody titer by ELISA at 10 to 14days following each injection. The antiserum collected at day 180,designated R399, was used for immunoprecipitation and radioimmunoassay.

Monoclonal Antibodies A monoclonal antibody against recombinant TGFα waskindly provided by Genentech Corp.

Measurement of Anti TGFα Antibody (R.399) Levels by ELISA

Micro-Elisa plates (Dynatech-Immunolon II, Dynatech Laboratories, Inc.Chantilly Va.) were coated for 16 hours at 4° C. with 500 ng/ml ofrecombinant TGFα in 50 mM sodium carbonate buffer (pH 9.6). The samplesto be assayed (antibody) were serially diluted 1:1,000-1:64,000 with0.15M NaCl, 0.05M Tris-HCl (pH 7.4), 2 mM EDTA, 5 mg/ml bovine serumalbumin, 0.05% Tween 20 (TBS-BSA-Tween) and were incubated in the wellsfor 2 hours at 37° C. The plates were washed five times with PBS-Tweenand then incubated for 1 hr at 37° C. with horseradishperoxidase-conjugated goat anti-rabbit immunoglobulin in TBS-BSA-Tween.The plates were then washed five times with PBS-Tween and incubated for4 hrs at 22° C. with 100 1 per well of 0.1 mg/ml o-phenylenediamine,0.012% H₂ O₂ in 0. 1M Phosphate-citrate buffer (pH 5.0). The reactionwas stopped by the addition of 50 1/well of 2.5N H₂ SO₄ and theabsorbance was measured at 492 nm using a UR 700 Microplate Reader(Dynatech Lab., Inc., Chantilly, Va.).

Radioimmunoassay (RIA)

TGFα RIA The presence of peptides immunologically related to TGFα wasdetermined using a RIA kit with a polyclonal anti-rat TGFα and rat ²⁵ I!TGFα (Biotope, Inc., Seattle, Wash.). This antibody does not cross-reactwith human EGF. Aliquots of conditioned media were reduced with 40 mMdithiothreitol and denatured by immersion for 1 minute in a boilingwater bath. Assays were done in duplicate according to themanufacturer's protocol and each collection of conditioned media wasassayed at least twice.

Solid Phase RIA 96 well microtiter plates were coated with anti-TGFαantibody (R399 or monoclonal antibody) for 2 hours at 37° C. The wellswere then filled with 100 l of the column fraction to be assayed forTGFα activity. A standard curve was constructed using 0.075 to 15 ngunlabelled TGFα. After the 2 hours incubation 5×10⁴ cpm of ¹²⁵ ! TFGα or2×10⁵ cpm of metabolically labelled antigen was added per well. Theplates were incubated further for 16 hours at 4° C. The wells were thenwashed and counted using a gamma counter (Model B5002, PackardInstruments Co., Sterling, Va.). The EGF RIA was performed with ananti-EGF antibody (Oncogene Science clone 144-8, Manhasset, N.Y.). Astandard curve was constructed using human EGF (hEGF, receptor grade,Collaborative Research, Waltham, Mass.).

Metabolically labelled TGFα-like material from MDA-MB-231 cells reactedonly with the polyclonal antibody. In contrast, the two antibodiescross-reacted with metabolically labelled material derived from H8 cellsand no immuno-reaction was noted with pre-immune serum (normal rabbitserum, NRS) or metabolically labelled conditioned media from Hs578tbreast carcinosarcoma cells (FIG. 1), which do not produce TGFα mRNA.Thus only the monoclonal antibody is able to distinguish between TGFαand the TGFα-like material from MDA-MB-231 cells. Specificity of theassay was demonstrated using a competition RIA with unlabelledrecombinant TGFα.

Labelled material from MDA-MB-23 1, H8, and Rat-FeSrV cells wasimmunoprecipitated with the anti-TGFα polyclonal antibody. ³⁵S!-labelled proteins released into the conditioned media by thedifferent cell lines were immunoprecipitated with 10 μg (specific ornon-specific) antibody partially purified by 45% ammonium sulfateprecipitation. After solubilization, the immuno-precipitates wereanalyzed by 15% SDS-PAGE and subsequent fluorography. Prestainedmolecular weight markers (Biorad, Richmond, Calif.) were run in parallellanes.

Detection of an immunoreactive species of approximately 30 kDa sizeverified the secretion of a higher molecular weight TGFα-likepolypeptide in MDA-MB-231 cells. H8 cells, which overexpress classicalTGFα, yielded a 6 kDa product. The expected 18 kDa precursor of theclassical 6 kDa TGFα was precipitated from Rat-FeSrV, which are known tosecrete the "normal" precursor. The intensity of the bands diminishedwhen the immunoprecipitation was performed in the presence of excessunlabelled TGFα. No specific bands were immunoprecipitated by pre-immunerabbit serum.

EXAMPLE 2 Identification of erbB-2 Ligands

A monoclonal antibody (4D5) radioreceptor assay was used to screenconditioned media derived from different human cell lines for thepresence of p185^(erbB-2) binding activity. Conditioned media wascollected as described by Bates, et al., Cancer Res. 46:1707-1713(1986). Briefly, media were concentrated 100-fold in an Amiconultrafiltration cell (YM5 membrane) (Amicon, Denvers, Mass.). Onceclarified and concentrated, the media were stored at -20° C. whileconsecutive collections were made during the following days. Theconcentrated media were dialyzed using Spectraphore 3 tubing (SpectralMedical Industries, Los Angeles, Calif.) against 100 volumes of 0.1Macetic acid over a two day period at 4° C. The material thatprecipitated during dialysis was removed by centrifugation at 400 rpmfor 30 min. at 4° C.; protease inhibitors were added as described byBates, et al., Cancer Res., 46:1707-1703 (1986).

A monoclonal antibody, 6E9, (Fendly, et al., Cancer Res., 50:1550-1558(1990)) against the extracellular domain of p185^(erbB-2) which does notcompete with gp30 for p185^(erbB-2) binding was used as a control, torule out the possibility that the erbB-2 oncoprotein or the portion ofit that is shed from the cells into their growth media might interferewith the 4D5 assay. Shed erbB-2 extracellular domain would compete withthe binding of both 4D5 and 6E9 to p185^(erbB-2), as opposed to a erbB-2ligand which would compete exclusively with 4D5. Any antibody which isdirected against the p185^(erbB-2) extracellular domain can be used as acontrol antibody in place of 6E9, provided that the control antibodydoes not interfere with 4D5 or M0193 binding, i.e., that the controlantibody and the test antibody do not bind the same binding site on theextracellular domain.

Binding assays were performed as described (Lupu, et al., Science, 249:1552 (1990)). Proliferating SK-Br-3 cells were incubated with iodinatedanti-erbB2-antibodies (4D5 or 6E9) in the presence of severalconcentrations of conditioned media. Media derived from SK-Br-3, MCF-7,HS578T, MDA-MB-453, BT-549, MDA-MB-468, and MDA-MB-157 breast cancercells, as well as several nonmalignant and transformed breast epithelialcells (Cell lines studied were non-malignant breast epithelial 184cells, immortalized 184A1N4 cells and oncogene-transformed 184A 1N4T(SV-40), 184A 1N4H (ras), 184A 1N4M (myc), 184A1N4TH (SV-40, myc), and184A1N4MH (myc, ras) cells were evaluated.) Conditioned media fromMDA-MB-231 cells were used as a positive control since these cells wereknown to secrete gp30. Only one of the 15 different media tested, thatfrom SK-Br-3 cells, showed an ability to compete with 4D5 forp185^(erbB-1) binding.

EXAMPLE 3 Purification of the TGFα-like Polypeptide

TGFα-like material was isolated from serum-free conditioned media ofMDA-MB-231 cells. Levels of TGFα-like polypeptide were quantified bythree independent assays: capacity to induce anchorage-independentgrowth of NRK fibroblasts in soft agar, ability to compete with ¹²⁵ I!EGF for EGF receptor binding on A431 human carcinoma cell membranes andcross-reactivity with polyclonal antibodies raised against mature TGFα.EGF receptor binding activity and TGFα immunoreactivity were detectedusing a RIA kit provided by Biotope.

EGF Radioreceptor Assay

A431 membranes were prepared according to the method of Kimball andWarner. A431 cells were disrupted under nitrogen and the nuclei andorganelles pelleted by low speed centrifugation. The membranes were thenpelleted by centrifugation at 35,000 rpm for 1 hour and resuspended in20 mM HEPES buffer, pH 7.4. Membranes (2.5 g/ml) were plated into 96well plates and allowed to dry overnight at 37° C. before use. Standardbinding competition studies were performed using ¹²⁵ I! EGF (ICN, CostaMesa, Calif., specific activity-100 Ci/g, about 50,000 CPM/well). Astandard curve was constructed with 0.075-10 ng of unlabelled HEGF(receptor grade, Collaborative Research). The different fractions to beanalyzed were lyophilized and reconstituted in PBS (0.5 ml/500 mlconditioned media). After incubation of the labelled EGF and 10 l of thesamples for 2 hours at 37° C. in binding buffer (IMEM containing 50 mMHEPES and 0.1% BSA pH 7.7), the wells were washed, cut from the plateand counted. EGF-competing activity, was computed using a HewlettPackard RIA Program.

Molecular Filtration Chromatography

To determine the approximate molecular weight of the MDA-MB-231 derivedTGFα-like polypeptide, 5 ml of 100-fold concentrated, dialyzedconditioned medium was chromatographed by gel filtration using SephadexG-100. Lyophilized conditioned medium was dissolved in 1M acetic acid toa final concentration of about 25 mg/ml total protein. Insolublematerial was removed by centrifugation at 10,000 rpm for 15 minutes. Thesample was then loaded onto a Sephadex G-100 column (XK 16, Pharmacia,Piscataway, N.J.), was equilibrated and was subjected to elution with 1Macetic acid at 4° C. with an upward flow of 30 ml/hr. 100 ng of proteinwas processed from 4 ml of 100-fold concentrated medium. Fractionscontaining 3 ml of eluate were lyophilized and resuspended in 300 l PBSfor assay and served as a source for further purification.

Elution was performed with 1.0M acetic acid and fractions werecharacterized for protein content. TGFα-like activities were eluted fromthe column in a single broad peak. Maximal activity was observed at anapparent molecular weight of 30 kDa and was separated from the bulk ofcontaminating proteins present in the bed volume. All the fractionsdemonstrating TGFα immunoreactivity also contained EGF receptor bindingactivity. The relative amounts of receptor binding activity andimmunoreactivity present in these fractions, however, appeared todiffer.

Heparin Affinity Chromatogaphy

Further analysis of the TGFα-like polypeptide from MDA-MB-231 cells wascarried out using heparin-sepharose affinity chromatography.Heparin-sepharose affinity chromatography was performed onunconcentrated conditioned media from MDA-MB-231 cells.

Media conditioned by MDA-MB-231 cells were clarified by centrifugationfor 20 minutes at 2,000 rpm at 4° C. The supernatant was collected andstored at -70° C. After allowing the heparin-sepharose (Pharmacia,Piscataway, N.J.) to expand in PBS, 2 ml of gel was loaded on an Econocolumn (Biorad, Richmond, Calif.) and washed with about 100 bead volumesof PBS. Conditioned media were run through the beads by gravity (flowrate 20 to 50 ml/hr). The gel was then washed with 5 volumes of PBS andeluted stepwise with an increasing gradient of NaCl in 10 mM Tris-HCl,pH 7.0 (elution buffer). Gradient steps of 0.4M, 1.1M, 2.0M and 3.0MNaCl were used in the elution buffer until the 280nm absorption duringeach step returned to baseline (usually 3 to 5 column bed volumes). Theeluate was desalted on G-25 columns (Pharmacia, Piscataway, N.J.) andfilter-sterilized before use in the different bioassays. Pooledfractions containing active materials were also desalted on PD10 columns(Pharmacia, Piscataway, N.J.) before running through HPLC and FPLC.

In all experiments, less than 20% of the TGFα activity loaded onto thecolumn was recovered in the unabsorbed fractions. A sharp peak of erbB-2phosphorylation activity and EGF receptor binding activity was eluted byheparin-sepharose chromatography at a concentration of 0.6-0.9M NaCl.This activity represented one major 30 kDa molecular weight protein,which retained 70%-80% of the load activity.

FIG. 1A illustrates the use of low affinity heparin chromatography. Inparticular, affinity chromatography of conditioned media from MDA-MB-231cells was performed on a heparin-sepharose column. Fractions wereanalyzed for EGF receptor binding activity of A43 1 cell membranes.Aliquots from the input media and from the fractions containing activitywere analyzed by a 15% SDS-PAGE, followed by silver staining. Lane 1shows unconcentrated conditioned media. Lane 2 represents the activefraction.

Reversed-Phase High Pressure Liquid Chromatography (HPLC)

The TGFα-like polypeptide (the erbB-2 ligand) was further purified byreversed phase chromatography (HPLC) in two steps. A pool of fractionscontaining EGF receptor-competing activity from heparin-sepharosechromatography was reconstituted in 0.05% TFA in water and thenchromatographed on a Bondapak C₃ column. A steep acetonitrile gradient(0-100%) was used in this step. TGFα-like polypeptide elutes as a sharppeak in 30% acetonitrile and is separated from the bulk of thecontaminating proteins.

Steep Acetonitrile Gradient Steep acetonitrile gradient and all otherHPLC steps were carried out at room temperature after equilibration ofthe C3-Reversed phase column with 0.05% TFA (Triflucroacetic acid) inwater (HPLC-grade). The samples were loaded and fractions eluted with alinear gradient (0-45% acetonitrile in 0.05% TFA) at a flow rate of 1ml/min over a 30 minute period. Absorbance was monitored at 280 nm. Oneml fractions were collected and lyophilized before analysis for EGFreceptor-competing activity. The capacity of the individual fractions tocompete for EGF receptor binding (as described above) and to stimulatethe growth of NRK cells in soft agar was determined.

Soft agar cloning assays (anchorage-independent growth assays) werecarried out using a 1 ml bottom layer of IMEM containing 0.6% Bacto-agar(Difco, Detroit, Mich.), 10% FBS, and 2 mM glutamine in 35mm tissuedishes (Costar, Cambridge, Mass.). A 0.8 ml top layer of IMEM containingthe test samples, 0.36% agar, 10% FBS, and 3×10⁴ NRK cells was addedafter solidification of the bottom layer. Each sample was plated intriplicate. All samples were sterilized by filtration using a 0.22 mMillex CU millipore filter before plating. Plates were incubated in ahumidified, 5% CO₂ atmosphere at 37° C. and were counted after 12 daysincubation with a Bausch and Lomb Stem Cell Colony Counter (ArtexSystems Corp, Farmingdale, N.Y.).

Shallow Acetonitrile Gradient A pool of the active fractions wasrechromatographed on the same column. Generally, fractions were elutedwith an 0-20% acetonitrile gradient in 0.05% TFA for 5 minutes followedby a linear 20-40% acetonitrile gradient. The TGFα-like polypeptideactivity was eluted at 25-30% acetonitrile and effectively separatedfrom other contaminant proteins.

In a particular experiment, the pool of active fractions from theprevious HPLC step was rechomatographed over the same column. Elutionwas performed with a 0-18% acetonitrile gradient in 0.05% TFA over a 5minute period followed by a linear 18-45% acetonitrile gradient in 0.05%TFA over a 30-minute period. The flow rate was 1.0 ml/min and 1 mlfractions were collected. Human TGFα-like factor was eluted at a 30-32%acetonitrile concentration as a single peak detectable by RRA.

FIG. 1B illustrates the use of reversed-phase chromatography. Notably,the EGF/TFGα-active fractions obtained after heparin-sepharosechromatography were chromatographed twice on a μBondapak C₃ column in0.05% TFA. Samples were eluted with a steep gradient of acetonitrile.Fractions that showed EGF receptor binding activity were thenrechromatographed and eluted with a shallow acetonitrile gradient. EGFcompeting activity was constantly eluted at a 25-30% acetonitrilegradient. The resulting fraction was analyzed on a 15% SDS-PAGE followedby silver staining. Sizes are shown in kilodaltons.

In order to achieve a complete separation of TGFα-like polypeptide fromthose impurities detected by silver staining (data not shown) we usedsize exclusion chromatography under acidic conditions. The activefractions for erbB-2 phosphorylation or EGF receptor-competing activitywere pooled and analyzed by SDS-PAGE. One single polypeptide band wasobserved after silver staining.

A summary of the steps leading to the isolation and purification ofTGFα-like polypeptide is presented in Table 1. A 27% recovery ofactivity and approximate 5400 fold purification was achieved.

                  TABLE 1    ______________________________________    Purification of TGFα-Like Activity    from Conditioned Medium from MDA-MB-231 Cells*                       EGF                       Compe-   Relative                       ting     Specific                                      Degree              Protein  Activity.sup.b                                Activity                                      of              Re-      (Units/  (Units/                                      Puri- Recovery    Purification              covered.sup.a                       mg       mg    fication                                            (%    Step      (mg)     TGFα)                                protein)                                      (fold)                                            Activity)    ______________________________________    Conditioned              98       450      4.6   1     100    Medium    Acid-Soluble              82       419      5.1   1     93    Supernatant    Gel Filtration              2.95     209      70.8  15.3  46    1-Heparin-              1.54     230      149   32.3  51    Sepharose    2-Reverse Phase.sup.c              0.03     173       5768 1253  38    3-Reverse Phase.sup.d              0.006    124      24800 5400  27    ______________________________________     .sup.a Total protein was determined using BSA as a standard. The     quantitation of step 6 was based on extrapolation from standard values.     The absolute specific activity of a comparison aliquot was found to be 1     million units/mg.     .sup.b One unit of EGF competing activity is defined as the amount of     protein that inhibits the binding of  .sup.125 I!EGF to the receptor by     50%.     .sup.c Steep acetonitrile gradient.     .sup.d Shallow acetonitrile gradient.     12-3: Subsequent purification steps     *Each value represents the mean of 4-6 experiments and they were     reproducible within 10%.

EXAMPLE 4 Characterization and Purification of the erbB-2 Ligand

Since SK-Br-3 cells seemed to secrete a erbB-2 ligand, our next step wasto determine whether the putative ligand showed heparin binding, whichwould be consistent with the secretion of gp30. No p185^(erbB-2) bindingactivity was detected after media from SK-Br-3 cells was processed byheparin chromatography, suggesting that either gp30 was not present inthe media or, since SK-Br-3 cells release p185^(erbB-) 2 extracellulardomain (ECD) into the culture media, that ECD might interfere withheparin binding or might be washed off the column bound to theextracellular domain.

Our next step was to develop a purification procedure which would notinterfere with p185^(erbB-2) ECD. Recombinant p185^(erbB-2)extracellular domain (ECD) (obtained from Genentech Inc., Calif.) wascoupled to a polyacrylamide-hydrazide sepharose affinity chromatographymatrix (Avromeas, et al., Scand. J. Immunol., 8:7 (1978)). The molecularweight of this extracellular domain is approximately 94 kilodalton.

erbB-2 extracellular domain (ECD) was coupled topolyacrylamide-hydrazide Sepharose beads (Sigma). After extensive washesof the beads (5 volumes) with ice-cold 1.0M HCl, the beads wereactivated with 0.5M NaNO₂ (1 volume). The temperature was maintained at0° C. for 15-20 minutes, then the beads were filtered on a sinteredglass funnel and washed with ice-cold 0.1M HCl. The beads wereimmediately washed with 0.1M sulphamic acid and then ice-cold water, andresuspended in 0.2M NaHCO₃, pH 6.0 (10 volumes).

The coupling was tested by demonstrating the binding of iodinated 4D5antibody to the column. Other antibodies such as M0193 can also be used.

The percent of EDC binding to the sepharose beads was between 90-98%.1.0 ml of gel was loaded on an Econo column (Biorad, Richmond, Calif.)and washed with about 100 bead volumes of PBS. After the column waspacked, conditioned media, derived from SK-Br-3 human breast cancercells, were run through the beads by gravity (flow rate 30 ml/hr). Thecolumn was then washed with 5 volumes of PBS and eluted stepwise with1.0M Citric Acid at different pHs (from 4.0 to 2.0). Usually 10 bedvolumes of each pH solution were employed. All fractions were desaltedon PD10 columns (Pharmacia, Piscataway, N.J.) before testing theirbiological activity. Aliquots from the input media and from thefractions containing activity were analyzed by a 10% SDS-PAGE, followedby silver staining.

Concentrated conditioned media from SK-Br-3 cells (clones 21, 22) wereloaded onto the column and elution of the material obtained wasperformed stepwise with 1.0M Citric Acid across a pH range from 4.0 to2.0, to allow the dissociation of the erbB-2 ECD and putative ligand.The fractions were tested for their p185^(erbB-2) binding properties inthe 4D5 binding assay.

A single purification yielded an apparent homogeneous polypeptide of 75kilodaltons at 3.0-3.5 elution pH. The homogeneity of the sample wasconfirmed by analysis on a SDS-PAGE (Laemmli U. K., Nature, 227:680(1970)) by silver staining (Morissey, J. H., Anal. Biochem., 177:307(1981)) (FIG. 7). Lane 3 shows unconcentrated conditioned media fromSK-Br-3 cells (clones 21, 22). Lane 2 represents the elution at pH 3.0,lane 1 represents the elution at pH 3.5. Sizes are shown in kilodaltons.This preparation was the source used for further experiments.

To confirm that the 75 kDa polypeptide (p75) obtained from the ECDaffinity column at pH 3.0-3.5 elution of SK-Br-3 conditioned media wasindeed a ligand for the erbB-2 oncoprotein we used two independentassays. We first confirmed, with the 4D5 radioreceptor assay, that p75(eluted at pH 3.0-3.5) binds specifically to the erbB-2 receptor inSK-Br-3 cells, while material from other chromatography fractions orflow-through did not show such activity (Table 2). The binding ofiodinated 6E9 antibody to p185^(erbB-2) was not altered by p75,suggesting that the eluted material was not p185^(erbB-2) ECD.

                  TABLE 2    ______________________________________    Binding of chromatography fractions from SKBr-3 cell conditioned    medium (p75) to p185.sup.erbB-2 and EGFR.                 % p185.sup.erbB-2                          % EGFR                 binding  binding    ______________________________________    Control        100        100    Flow-Through   99         96    pH 2           99         95    pH 2.5         91         95    pH 3           9          95    pH 3.5         18         99    pH 4           63         98    ______________________________________     Sk-Br-3 and MDAMB-468 cells were plated (100,000 cells/well) in 24well     plates in 5% FCS IMEM (Biofluids). Binding studies were performed as     described (Lupu, et al., Science 249: 1552-1555 (1990)). 50 ml of 100x     conditioned media from SKBr-3 cells were loaded into a p185.sup.erbB2     extracellular domain affinity chromatography column. Flowthrough and     fractions eluted with 1 citric acid (pH gradient from 4 to 2) were     collected. After neutralization (pH 7.4) and desalting with PBS, the     fractions were tested for p185.sup.erbB2 and EGFR binding activity.     Iodinated 4D5 antibody was used to assess p185.sup.erbB2 binding in SKBr-     cells, and iodinated EGF was used to assess EGFR binding in MDAMB-468     cells. Results are shown as percent of control (no treatment) binding.     Each experiment was performed in triplicate, and the SD were less than 15     in all cases.

Since gp30 had been identified as a ligand common to both the EGFR andp185^(erbB-2), we also tested the activity of all the eluted fractionsfrom SK-Br-3 conditioned media in a EGFR binding assay using MDA-MB-468cells and iodinated EGF. In this assay EGF and gp30, used as controls,displaced the binding of iodinated EGF in a dose dependent manner. Incontrast, none of the eluted fractions derived from SK-Br-3 conditionedmedia showed activity, indicating that p75 does not bind to the EGFR(Table 2).

EXAMPLE 5 The TGFα-like Polypeptide (erbB-2 Ligand) Is Glycosylated

The apparent heterogeneity in size of the larger TFGα species and thepotential for N-linked glycosylation of the TFGα precursor at Asn 25 ledto the consideration of whether the high molecular weight TGFα-likepolypeptide secreted from MDA-MB-231 cells was a glycosylated form ofTGFα.

Tunicamycin Treatment

Tunicamycin (Sigma, St. Louis, Mo.) was dissolved in 50 mm sodiumcarbonate (pH 10.0) and filter-sterilized with a 0.22 m filter.Confluent monolayers of MDA-MB-231, MCF-7 and Hs578T cells were grown inIMEM in the presence of 20 g/ml tunicamycin (unless otherwise specified)for 4 hours prior to metabolic labelling. Metabolic labelling was thenperformed as described above with continued tunicamycin treatment.

When MDA-MB-23 1 cells were incubated with tunicamycin, an inhibitor ofco-translational N-linked glycosylation, and the media wasimmunoprecipitated with the anti-TGFα polyclonal antibody, a species of22 kDa replaced the previously observed 30 kDa species.

These proteins were subjected to enzymatic treatment with N-glycanaseand elastase.

Elastase Treatment

The samples containing TGFα-like activity were incubated with 20 gporcine pancreatic elastase (Sigma) dissolved in 50 mM glycylglycine, pH7.9, for 1 hour at 22° C. The samples were then subjected toimmunoprecipitation and SDS-PAGE analysis.

Additional cleavage of the 22 kDa polypeptide with elastase yielded anapparent 11 kDa product, different from the mature 6 kDa TGFα that wasobserved in Rat-FeSrV labelled media. The 11 kDa product had a higherimmunoreactivity with the R399 antibody than the 30 kDa and the 22 kDapolypeptides. Shorter exposure of the gel showed clearly a precipitatedband near the 11 kDa molecular weight. Tunicamycin treatment did notsignificantly affect the levels of secreted TGFα activity as determinedby both RIA and EGF receptor binding assays.

N-Glycanase Digestion

The purified 30 kDa TGFα-like protein was subjected to digestion withN-glycanase. Samples equivalent to 100 ng were incubated with 50 1 of0.2M sodium phosphate (pH 8.6), 1.25% NP40 and 2-6 g N-glycanase(Genzyme Corp., Boston, Mass.) were subsequently added to each sampleand incubated at 37° C. for 16 hours. 50 μl of 3-fold concentratedloading buffer was added before electrophoretic analysis, performed asoutlined above. The gel was silver stained.

When the purified 30 kDa polypeptide was treated with N-glycanase, a 22kDa product was detected by silver staining. The absence of cleavage ofthe purified 30 kDa polypeptide after 0-glycanase treatment suggeststhat no O-glycosylation occurs in this system.

EXAMPLE 6 Peptide Mapping

In order to determine the degree of homology between the novel 30 kDaTGFα-like growth factor and mature TGFα, peptide mapping was performedusing the method of Clevand. Immunoprecipitation of metabolicallylabelled conditioned media from MDA-MB-231, H8, and Rat-FeSrV cells wascarried out with the R399 anti-TGFα polyclonal antibody. Precipitateswere analyzed on SDS-PAGE and the specific bands were electroeluted (30kDa from MDA-MB-231 cells, 6 kDa from H8 cells, and 18 kDa from theRat-FeSrV cells).

Electrophoretic Elution of Radiolabelled Protein from Gels

After fluorography of an SDS-PAGE, bands of interest were excised andthe protein eluted by electrophoresis into a dialysis tubing over 16 hrsat 120 volts. The contents of the dialysis bag were cooled at 4 C. andthen precipitated by the addition of trichloroacetic acid to a finalconcentration of 20%. The precipitates were pelleted by centrifugation,washed twice with ethyl ether, and resuspended in loading buffer.

Digestion Procedure for Purified Eluted Proteins

Electroeluted proteins were dissolved at approximately 0.5 mg/ml inloading buffer which contained 0.125M Tris-HCl (pH 6.8), 0.5% SDS, 10%Glycerol and 0.001% Bromophenol Blue. The samples were then heated at100, C. for 5 minutes. Proteolytic digestion were carried out at 37° C.for 30 minutes by the addition of Staphylococcus aureus Protease V8(Sigma, St. Louis, Mo.) to a final concentration of 25 g/ml according tomethods. O-mercaptoethanol and SDS were subsequently added to finalconcentrations of 20% and 2%, respectively. Proteolysis was stopped byboiling for 2 min. The samples were then injected on a C18 ReversedPhase HPLC column.

The products were then subjected to a peptide digestion using 25 g/mlV8-protease. After complete digestion, the samples were analyzed by C18reversed phase chromatography. Three major peptide peaks eluted atdifferent acetonitrile concentrations by reversed phase chromatography.However, the concentrations at which those peptides isolated fromMDA-MB-231 cells eluted (16%, 18.7%, and 21.7%) were different from thepeptides isolated from H8 and FeSrV cells (24%, 29%, and 32.6%). Thepeptide elution pattern of the TGFα (6 kDa) derived from H8 cells andRat-FeSrV cells was essential identical. The same results were obtainedwith 40 g of V8 protease, indicating that concentration of the enzymewas not responsible for the differential peptide cleavage.

RNA Extraction

Moreover, in vitro translation of mRNA derived from MDA-MB-231 cells andH8 cells was done. Total cellular RNA was extracted from cells byhomogenizing in guanidine isothiocyanate followed by centrifugation overa cesium chloride cushion. Poly (A)⁺ mRNA was eluted in 10 mM Tris afterpassing total cellular RNA over an oligo (dT) cellulose column(Pharmacia, Piscataway, N.J.) equilibrated with 10 mM Tris-0.5M NaCl pH8.0. After precipitation in ethanol (66% vol/vol) and 0.1M acetic acid,both total and poly(A)⁺ selected RNA were resuspended in 10 m Tris-1 mMEDTA buffer and separated on 1% agarose, 6% formaldehyde gels.Electrophoresis was carried out at 20 volts over 14-16 hours in: 5 mMNaAc 1 mM EDTA, 20 mM 3- N-morpholino! propane sulfonic acid pH 7.0(MOPS-Sigma). The gels were stained with ethidium bromide 2.0g/ml toallow inspection of the quality and quantity of RNA (). In vitrotranslation assays were performed using Wheat Germ kit according to themanufacturer's instructions (Promega).

The resulting polypeptide had the same peptide mapping profile than thepurified 30 kDa factor after treatment with N-glycanase and elastase.These results provide evidence that a precursor different than the"normal" TGFα precursor is translated from the mRNA of MDA-MB-231 cells.Moreover, the above results indicate that the MDA-MB-231 derivedTGFα-like polypeptide shares very few, if any, common peptide sequenceswith mature TGFα.

EXAMPLE 7 Receptor Binding Activity

The EGF receptor binding activity of the 30 kDa TGFα-like protein wascompared with that of EGF in a radioreceptor assay. Both growth factorscompeted with ¹²⁵ I! EGF for receptor sites on A431 membranes. Thespecific EGF-competing activity of the purified TGFα-like polypeptidewas found to be 1-1.5×10⁶ units/mg; 1.1 ng of TGFα-like polypeptide wasrequired to inhibit EGF binding by 50%. TGFα-like polypeptide was aseffective as EGF in EGF receptor binding.

p185^(erbB-2) receptor binding was studied by competition assay inSK-Br-3 cells. SK-Br-3 cells were plated in 24 well plates in IMEM(Biofluids) supplemented with 5% FCS. After a wash with binding buffer(DMEM/F12 pH 7.4, containing 1 mg/ml BSA, 10 Mn hepes and 20 Mmglutamine) cells were incubated for 30 minutes at 37° C. with bindingbuffer. The EGFR were saturated with 30 nM EGF for 2 hours at 4° C. p185binding studies were then performed for 3 hours at 4° C. with 1 nMiodinated 4D5 in the presence of various concentrations of unlabeledgp30 or 4D5. After the incubation, cells were washed 3 times withbinding buffer and then solubilized with 1% SDS. The results are shownin FIG. 5. No specific binding was determined with excess (100 nM) ofunlabeled antibody. Each group was assayed in triplicate. Theexperiments were performed five times and the results were reproducible.

EXAMPLE 8 Biological Characterization of the TGFα-like Material

In order to characterize the cellular effects of the present 30 kDaglycoprotein, various experiments were conducted. The purified 30 kDaTGFα-like polypeptide stimulated the growth of serum NRK fibroblasts andinduced colony formation of these cells in soft agar using the proceduretaught in Example 3.

FIG. 12 illustrates the effect of gp30 on soft agar colony formation ofSK-Br-3 cells.

FIG. 13 illustrates the effect of gp30 on soft agar colony formation ofMDA-MB-468 cells.

FIG. 14 illustrates the effect of gp30 on soft agar colony formation ofMCF-7 cells.

FIG. 15 illustrates the effect of EGF on soft agar colony formation ofSK-Br-3 cells.

FIG. 16 illustrates the effect of EGF on soft agar colony formation ofMDA-MB-468 cells.

The bioactivity of the purified TGFα-like polypeptide was also tested byanchorage-dependent growth assays of the carcinogen-immortalized humanmammary epithelial cells 184A1N4 and anchorage-independent growth assaysof 184A1N4-derived cells partially transformed by SV40 T antigen,184A1N4T.

Anchorage-Dependent Growth Assay

Cells were grown in IMEM containing 5% FCS. Upon confluence cells weredetached using trypsin-versene (Biofluids, Rockville Md.) and passed at1:20 to 1:50 dilutions. Cells were seeded in 12-well plates at4,000-10,000 cells/well, depending on the cell type (MDA-MB-231-8,000cells/well in serum free IMEM). After 24 hours the media was changed andthe cells were treated with EGF, TGFα or TGFα-like protein wereharvested at 1, 2 and 4 days using trypsin-versene. The cells werecounted using a coulter counter. Dose response curves of TGFα-likepolypeptide on these cells were similar to those observed with EGF andTGFα.

EXAMPLE 9

The biological activity of the purified 30 kDa TGFα-like factor wasfurther assessed by examining its ability to induce autophosphorylationof the EGF receptor. A431 cells, which overexpress the EGF receptor,were incubated with various concentrations of EGF, TGFα or TGFα-likegrowth factors. Each of the three peptides similarly stimulatedphosphorylation of the EGF receptor.

Phosphorylation of the EGF Receptor

Subconfluent A431 cells were cultured in IMEM for 10-12 hours. The cellswere treated with 10-30 nM TGFα, EGF or TFGα-like growth factor for 30minutes at 37° C. Cells were lysed in 20 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1% NP40, 1 mM EDTA, 2 mM PMSF, 42 mM leupeptin andimmunoprecipitated as described above using monoclonal antibody 225directed against the EGF receptor (Oncogene Science, Manhasset, N.Y.).The immuno-precipitates were washed three times with RIPA buffer andresuspended in 40 1 TNE (0.O1M Tris-HC1, pH 7.5, 0.15M NaCl, 1 mM EDTA).Five Ci of y_³² P! ATP was added to the immuno-precipitates and thetotal ATP concentration was adjusted to 15 mM (final) in a volume of 60l. The reaction mixture was incubated for 5 minutes on ice beforeaddition of 20 1 of 3×sample buffer. The samples were boiled for 5minutes and analyzed by denaturing 7.5% SDS-PAGE.

EXAMPLE 10

In order to characterize the cellular effects of the present 30 kDaglycoprotein ligand, its induction of tyrosine phosphorylation wasassessed in the human breast cancer lines MDA-MB-468 and SK-Br-3.Notably, MDA-MB-468 cells have amplification and over expression of theEGFR gene and do not express erbB-2 receptor-like protein. SK-Br-3 cellshave amplification and over expression of the erbB-2 gene as well asrelatively elevated levels of EGFR.

Detection of Phosphorylated Proteins in SK-Br-3 Cells

SK-Br-3 cells were grown in 90% confluence in 24-well plates (Costar).Cells were treated at 30° C. with IMEM (FIG. 2, lanes 1 and 2), IMEMcontaining 25 nb/ml recombinant TGFα (Genetech, California) (FIG. 2,lanes 3 and 4), and IMEM containing 5 ng/mi of gp30 (FIG. 2, lanes 5 and6), all of these in the presence (FIG. 2, lanes 1, 4, 5) and the absence(FIG. 2, lanes 2, 3, 6) of an anti-EGF receptor blocking antibody(Genetech, California). After 20 minutes the media was removed and cellswere lysed in 100 μl of sample buffer containing 1% SDS, 0.1%β-mercaptoethanol, 0.15M Tris-HCl (pH 6.8), 10% glycerol, 0.02%bromophenol-blue, 1 mM EDTA, 2 mM PMSF and 42 mM leupeptin. After 5minutes at 95° C., 50 μg of protein were loaded in a 7.5% SDS-PAGE.Proteins were then transferred to nitrocellulose membrane forimmunoblotting (Hoefer Scientific Instruments, California) byelectrophoresis in a modified method of Towbin, et al., using anelectrophoretic transfer unit (Hoefer, TE 22). Electrophoretic transferwas carried out at room temperature for one hour at 125 mA in a buffercontaining 25 Mm glycine, 129 Mm Tris (Ph 8.3) and 20% methanol.Following transfer, the filter was blocked with 5% BSA in Tris-BufferedSaline containing 0.5% Tween 20. An antiphosphotryosine antibody(Amersham) was reacted with the immobilized proteins in 5% BSA (SigmaRIA Grade). Immunocomplexes were detected by a goat anti-mouse antibodyconjugated to alkaline phosphatase. Blots were then incubated with acolor development substrate solution containing NBT and BCIP (Promega).

Alternatively, cells were grown to 80% confluence in a 35 mm dish(Costar). FCS was removed 16 hours prior to the labelling. Cells wererinsed with PO₄ ! free DMEM (GIBCO) and then incubated for 3 hrs at 37°C. with 1.0 mCi/ml of ³² Pi!/dish (32-orthophosphate Amersham). After 3hrs, cells were treated for 20 minutes at 37° C. with different samples(which includes gp30). Following the incubation the culture dishes wereplaced over an ice-bath and the cells were washed twice with PBS.Lysates were prepared with a modified Ripa buffer, containing 1% TritonX100, kinase, protease and phosphatase inhibitors, at 4° C. The celllysate was centrifuged at 10,000 ×g for 15 minutes at 4° C. Thesupernatant was incubated with 10 μl of normal mouse immunoglobulin for1 hr at 4° C., and the nonspecific complexes were clarified usingprotein A-sepharose (Sigma). The supernatant was incubated with amonoclonal anti-phosphotyrosine antibody IG2 (Kindly provided byFrackelton A. R.) and specifically eluted using 1 mM phenylphosphate. Asecond immunoprecipitation was then performed using a polyclonalantibody against the erbB-2 C-Terminal sequence or with a polyclonalantibody against the EGFR (Oncogene Science, New York). The specificcomplexes were precipitated with 5 mg/sample protein A-sepharose (Sigma)and the pellets were washed three times with lysis buffer and the pelletwas then resuspended in 50 μl sample buffer (50 mM Tris-HCl (pH 6.8), 2%SDS, 10% Glycerol, 0.1% bromophenol blue and 5% beta-mercaptoethanol).After 5 minutes at 95° C., the samples were loaded onto a 7.5% SDS-PAGE.

Phosphoaminoacid analysis

Phosphoproteins in individual bands were extracted from polyacrylaminidegels and then subjected to partial acid hydrolysis and two dimensionalthin-layer electrophoresis using HTLE-7000 (CBS Scientific C.O.), usinga well known procedure.

The 30 kDa ligand, TGFα and EGF were found to induce tyrosinephosphorylation in both cell lines, and EGFR blocking antibody abolishedthe phosphorylation induced by the three growth factors in MDA-MB-468cells. This antibody did not, however, completely block thephosphorylation induced by the present 30 kDa ligand in SK-Br-3 cells.However, it did block the phosphorylation induced by TGFα.

From the above result, it appears that tyrosine phosphorylation of aprotein is different from EGFR occurs in SK-Br-3 cells treated with 30kDa factor. No phosphorylation was observed in untreated SK-Br-3 cells,and cells treated with the anti-EGFR antibody alone.

EXAMPLE 11 Detection of Phosphorylated Proteins in MDA-MB-453 Cells

MDA-MB-453 cells were grown to 90% confluence in 24-well plates (Costar)and treated at 37° C. with IMEM (FIG. 3, lane 1), IMEM containing 25ng/ml of recombinant TGFμ (Genetech, California) (FIG. 3, lane 10), orIMEM containing 1.25-40 ng/ml of gp30 (FIG. 3, lanes 2-9). After 20minutes media was removed and cells were lysed in 100 μl of samplebuffer as described in Example 10. After 5 minutes at 95° C., 50 μg ofprotein was loaded in a 7.5% SDS-PAGE. Proteins were then transferred tonitrocellulose membrane for immunoblotting with an antiphosphotryosineantibody (Amersham) as described in Example 10.

In human mammary carcinoma cell line MDA-MB-453, which over expresseserbB-2, but which has undetectable levels of the EGF receptor protein ormRNA, the 30 kDa ligand was observed to induce a significant increase intyrosine phosphorylation in a dose dependent manner at concentrationsranging from 1.25 mg/ml to 50 mg/ml. By contrast, EGF and TGFα wereunable to induce tyrosine phosphorylation in the 185 kDa range, at aconcentration of 25 mg/ml. No phosphorylation was observed in untreatedcells. Hence, from the above, a direct interaction between the 30 kDaligand and the 185 kDa glycoprotein appears to occur.

EXAMPLE 12 Phosphorylation of p185 Protein in Intact CHO/DHFR andCHO/erbB-2 Cells

Cells were grown to 90% confluence in 24-2311 plates (Costar) in ═MEM(Biofluids) supplemented with 10% dialyzed FCS, 0.75 mg/ml G418, andMethotrexate (MTX) at concentrations of 50 nM (CHO parental andCHO-DHFR) or 250 nM (CHO-erbB-2). CHO-DHFR (FIG. 4A) and CHO-erbB-2(FIG. 4B) cells, were treated at 37° C. with control media supplementedwith 20 Mm Hepes (pH 7.4) (FIG. 4A and B, lanes 1 and 4), with 10 ng/mlof recombinant TGFα (Genetech, California) (FIG. 4A and B, lanes 2 and5), and control media supplemented 2.0 ng/ml of gp30 (FIG. 4A and B,lanes 3 and 6). After 20 minutes, media was removed and cells were lysedin 100 μl of sample buffer (as described in Example 10). Ananti-phosphotyrosine antibody (FIG. 4A and B, lanes 1 to 3) (Amersham)and an anti-erbB-2 antibody (FIG. 4A and B, lanes 4 to 6) (NEN), werereacted with the immobilized proteins in 5% BSA (Sigma RIA Grade).Immunocomplexes were detected as described for Example 10.

EXAMPLE 13 Inhibition of P185 Cross-linking With 4D5 Antibody of gp30

The binding assays were performed as described in Example 5. Binding wasperformed with iodinated 4D5 (1 nm) alone (FIG. 6, lane 1), in thepresence of 100 nM unlabeled 4D5 (FIG. 6 lane 2) and in the presence of2nM gp30 (FIG. 6, lane 3). 100 nM EGF were used as a control (FIG. 6,lane 4). Cells were then treated with a cross-linking agent EGS for 45minutes at 4° C., then quenched by adding 0.1 ml of 20 mM NH₄ Cl. Thesolubilized cells were immunoprecipitated with a polyclonal antibody tothe C-terminal domain of erbB-2 (Genetech, California). The precipitateswere analyzed on a 5% SDS-PAGE.

EXAMPLE 14

In order to determine the effects of the present 30 kDa ligand on theproliferation in colony formation of breast carcinoma cell lines, thefollowing experiment was conducted.

Cells were treated with the present 30 kDa growth factor, EGF, TGFα andanti-erbB-2 antibody in order to inhibit the proliferation of SK-Br-3cells.

It was observed that the anti-erbB-2 antibody inhibited theproliferation of the SK-Br-3 and MDA-MB-453 cells by 60-70% but did notinhibit the proliferation of MDA-MB-468 cells. Surprisingly, by exposingSK-Br-3, MDA-MB-453 and MDA-MB-468 cells to the 30 kDa ligand protein ofthe present invention, a 60-70% inhibition of cell growth was observedfor all cell lines.

Inhibition of growth by the 30 kDa ligand protein was reversed by anEGFR blocking antibody in MDA-MB-468 cells, but not in SK-Br-3 orMDA-MB-453 cells. This is an indication that the effects of the 30 kDaprotein on SK-Br-3 and MDA-MB-453 cells are not mediated through EGFR.

By contrast, the present 30 kDa glycoprotein exhibited no effect onMCF-7 cells, which have normal levels of EGFR and erbB-2. Additionally,EGF and TGFα inhibited the anchorage dependant growth of MDA-MB-468cells and SK-Br-3 cells, but not that of MDA-MB-453 or MCF-7 cells.EGF-induced anchorage dependant growth inhibition of SK-Br-3 andMDA-MB-468 cells was reversed by an anti-EGFR blocking antibody. In thepresence of the 30 kDa glycoprotein, the growth inhibition of SK-Br-3,MDA-MB-453 and MDA-MB-468 cells was nearly complete.

The growth inhibitory property of the present 30 kDa ligand appears tobe similar to that described for EGF on cells which express EGFR such asA431 cells and MDA-MB-468 cells.

EXAMPLE 15

Further, the growth of CHO/erbB-2 transfected cells was inhibited by70-80% after treatment with the present 30 kDa glycoprotein. No effectwas observed on the CHO/DHFR control transfectants and the parenteralCHO line. TGFα at the same molar concentration did not exhibit anyeffect on the proliferation of any of the three lines. Tyrosinephosphorylation and cell proliferation of the CHO/DHFR cells and theparenteral CHO cell line is not effected after treatments by the present30 kDa ligand or TGFα.

EXAMPLE 16 Cell Growth Inhibition by gp30

Sk-Br-3, MDA-MB-453, MDA-MB-468 and MCF-7 cells were plated in 24 wellplates in IMEM (Biofluids) supplemented with 5% FCS. Parental CHO cells,and CHO cells transfected with the DHFR gene or the erbB-2 gene wereplated in 24 well plates (Costar) in α-MEM (Biofluids) supplemented by10% dialyzed FCS, 0.75 mg/ml G418 and Methotrexate (MTX) 50 nM for theCHO parental and CHO-DHFR CELLS for 250 nm for the CHO-erbB-2. After 24hours media was removed and replaced with control serum free media (SFM)containing fibronectin, transferrin, hepes, glutamine, trace elements,and BSA, or SFM with the addition of 2.0 ng/ml gp30, 10 ng/mlrecombinant TGFα (Genetech), or with 2.5 μg/ml 4D5 specificanti-p185^(erbB-2) monoclonal antibody. Cells were grown in 90%confluence of control and counted. Each group was assayed in triplicate.The results are shown below in Table 3 as growth relative to control.The experiments were performed three times and the results werereproducible.

                  TABLE 3    ______________________________________                 MDA-    CHO/    CHO/  MDA-    SK-BR-3      MB-453  erbB-2  DHFR  MB-468                                             MCF-7    ______________________________________    gp30    31       24      20    99    18    100    4D5     32       34      22    98    104   92    antibody    TGFα            73       91      89    95    79    105    Control 87       91      87    94    92    99    antibody    ______________________________________

EXAMPLE 17

Since p75 interacted with the erbB-2 oncogene product, we next exploredwhether p75 activated p185^(erbB-2) We studied the ability of p75 tophosphorylate p185^(erbB-2) using MDA-MB-453 human breast cancer cells,which overexpress the erbB-2 oncoprotein but do not express detectablelevels of EGFR. First we determined that p75 activated tyrosinephosphorylation in MDA-MB-453 cells, using an anitphosphotyrosinemonoclonal antibody in a Western blot analysis (Towbin, et al., Proc.Natl. Acad. Sci. USA, 76:4350 (1979)) (FIG. 8A).

MDA-MB-453 cells were growth to 90% confluence in 24-well plate(Costar). Cells were treated for 20 minutes at 37° C. with control mediacontaining 20 mM Hepes, pH 7.4 (lane 1), control media containing 5ng/ml of gp30 (lanes 2), control media containing 4 ng/ml p75 (lane 3),control media containing 8 ng/ml p75 (lane 4), and control mediacontaining 2 ng/ml p75 (lane 5). The media was removed and cells werelysed in 100 μl of sample buffer containing 1% SDS, 0.1%O-mercaptoethanol, 0.15M Tris-HCl (pH 6.8), 10% glycerol, 0.02%bromophenol-blue, 1 mM EDTA, 2 mM phenylmethyl sulphonyl fluoride (PMSF)and 24 mM leupeptin. After 5 minutes at 95° C., 50 μg of protein wereloaded in a 7.5% SDS-PAGE. Proteins were then transferred to anitrocellulose membrane for immunoblotting (Heofer ScientificInstruments, California) by electrophoresis in a modified method ofTowbin, et al., PNAS, 76:4350 (1987), using a Hoeffer electrophoretictransfer unit (Hoeffer, Model number TE 22). Electrophoretic transferwas carried out at room temperature for one hour at 125 mA in a buffercontaining 25 mM glycine, 129 mM Tris (pH 8.3) and 20% methanol.Following transfer, the filter was blocked with 5% BSA in Tris-BufferedSaline containing 0.5% Tween 20. An antiphosphotyrosine antibody(Amersham) reacted with the immobilized proteins in 5% BSA (Sigma RIAGrade). Immune-complexes were detected by a goat anti-mouse antibodyconjugated to alkaline phosphatase. The blots were then incubated with acolor development substrate solution containing NBT and BCIP (Promega).

The specificity of the tyrosine phosphorylation for p185^(erbB-2) wasconfirmed by labeling MDA-MB-453 cells with ³² p! andimmunoprecipitating the p185^(erbB-2) oncoprotein with a polyclonalantibody that reacts with the C-terminal domain of p185^(erbB-2) butdoes not show cross-reactivity with EGFR (Hudziak, et al., Proc. Natl.Acad. Sci. USA, 84:7159 (1987)) (FIG. 8B). MDA-MB-453 cells were growthto 80% confluence in 24-well plates (Costar). The cells were washed thentwice with PO₄ free culture media PO₄ ! free MEM (GIBCO), with 10% PO₄free dialyzed fetal calf serum (FCS) and 2 mM glutamine. The cells werethen grown in 3 ml/well of PO₄ free culture media for 24 hrs.,subsequently the column of medium was adjusted to 1.0 ml/well and 0.5mCi of ³² Pi!/well was added and the cells were incubated for 6 hrs.

Cells were treated for 20 minutes at 37° C. with control media (lanes1), control media containing 2.0 ng/ml of gp30 (lanes 2), control mediacontaining 4.0 ng/ml p75 (lane 3), control media containing 8.0 ng/mlp75 (lane 4), and control media containing 2.0 ng/ml p75 (lane 5).Following the incubation, the culture dishes were placed over ice-bathand the cells were washed twice with PBS at 4° C. for 10 minutes with200 μl/well lysis buffer (50 mM Hepes, 150 mM NaCl, 1.0% Triton x-100, 1mM EGTA, 5 mM EDTA, 10% glycerol, 0.2 mM sodium orthovanadate, 0.5 mMPMSF, 20 μg/ml leupeptin, and 5 mM ATP, final pH 7.5). The cell lysatewas centrifuged at 10,000 g for 1 minute at 4° C. The supernatant wasincubated with 10 μl of normal rabbit serum (NRS) for 1 hr at 4° C. andthe non specific complexes were clarified using protein A-sepharose(Sigma). The supernatant was incubated with a polyclonal antibodyagainst the erbB-2 C-terminal sequence (Hudziak, et al., Proc. Natl.Acad. Sci. USA, 84:7159, 1987). The specific complexes were precipitatedwith protein A-sepharose (Sigma) and the pellets were washed three timeswith lysis buffer and the pellet was then resuspended in 50 μl samplebuffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromophenolblue and 5% beta-mercaptoethanol). After 5 minutes at 95° C., thesamples were loaded in a 7.5% SDS-PAGE. No phosphorylation was observedwhen MDA-MB-453 cells were treated with EGF.

In control experiments, phosphorylated EGFR was precipitated fromMDA-MB-468 cells with an anti-EGFR antibody after treatment with gp30,EGF and TGFα. MDA-MB-468 cells were grown to 80% confluence in 24-wellplate (Costar). The cells were washed twice with PO₄ free culture mediaPO₄ ! free MEM (GIBCO), with 10% PO₄ free dialyzed fetal calf serum(FCS) and 2 mM glutamine. The cells were then grown in 3 ml/well of PO₄free culture media for 24 hrs. Subsequently the volume of medium wasadjusted to 1.0 ml/well and 0.5 mCi of ³² Pi!/well was added and thecells were incubated for 6 hrs.

Cells were treated for 20 minutes at 37° C. with control media (FIG. 8C,lane 2), control media containing 10.0 ng/ml of p75 (FIG. 8C, lane 1),control media containing 4.0 ng/ml TGFα (FIG. 8C, lane 3), control mediacontaining 4.0 ng/ml EGF (FIG. 8C, lane 4), and control media containing2.0 ng/ml gp30 (FIG. 8C, lane 5). Following the incubation the culturedishes were placed over an ice-bath and the cells were washed twice withPBS at 4° C. Then, cells were lysed at 4° C. for 10 minutes with 200μl/well lysis buffer (50 mM Hepes, 150 mM NaCl, 1.0% Triton X-100, 1 mMEGTA, 5 mM EDTA, 10% glycerol, 0.2 mM sodium orthovanadate, 0.5 mM PMSF,20 μg/ml leupeptin, and 5 mM ATP, final pH 7.5). The cell lysate wascentrifuged at 10,000 g for 1 minute at 4° C.

The supernatant was incubated with 10 μl of normal rabbit serum (NRS)for 1 hr at 4° C. and the non specific complexes were clarified usingprotein A-sepharose (Sigma). The supernatant was incubated with apolyclonal antibody against the EGFR (Oncogene Science, New York). Thespecific complexes were precipitated with protein A-sepharose (Sigma)and the pellets were washed three times with lysis buffer and the pelletwas then resuspended in 50 μl sample buffer (50 mM Tris-HCl (pH 6.8), 2%SDS, 10% Glycerol, 0.1% bromophenol blue and 5% beta-mercaptoethanol).After 5 minutes at 95° C., the samples were loaded in a 7.5% SDS-PAGE.

p75 did not induce phosphorylation of the EGFR in these cells (FIG. 8C).These observations supported the hypothesis of an exclusive interactionbetween p75 and its receptor p185^(erbB-2).

EXAMPLE 18 Cellular Response to erbB-2 Ligand

We examined the biological effects of p75 in breast cancer cells, usinganchorage-dependent and anchorage-independent growth assays. SK-Br-3 andMDA-MB-468 cells were plated (30,000 cells/well) in 24 well plates inIMEM (Biofluids) supplemented with 5% FCS. After 24 hrs the media wasremoved and replaced with control serum free media containingfibronectin, transferrin, hepes, glutamine, trace elements, and BSA(SFM+), or SFM+ with the addition of purified p75 (4 ng/ml), purifiedgp30 (2.0 ng/ml) or with EGF (10 ng/ml). Cells were grown to 90%confluence of control and counted. Each group was assayed in triplicate.Results are shown as growth relative to control. The experiments wereperformed three times and the results were reproducible.

p75 inhibited the cellular proliferation of the overexpressing cellsSK-Br-3, BT-474 and MDA-MB-453 by 70-80% at a concentration of 4 ng/ml(The concentration of p75 polypeptide was determined by 4D5 bindingassay). No inhibition was observed in MDA-MB-468 cells, whichoverexpress the EGFR, or in MCF-7 cells which do not overexpressp185^(erbB-2) or EGFR. gp30, used as control, inhibited theproliferation of SK-Br-3 and MDA-MB-468 cells (FIG. 9). In ananchorage-independent growth assay, p75 inhibited the soft agar colonyformation of SK-Br-3 and MDA-MB-453 cells by 60-70%.

Since our initial experiments p75 inhibited the growth of breast cancercells, we speculated that either p75 was an inhibitory ligand or theinhibitory function of p75 resulted from hyperstimulation of the erbB-2receptor. SK-BR-3 cells were plated in 35 mm tissue culture dishes(Costar, Cambridge, Mass.). A bottom layer of 1.0 ml IMEM (Biofluids)containing 0.6% agar and 10% fetal calf serum (FCS) was prepared. Afterthe bottom layer was solidified, 10,000 cells per dish were added in a0.8 ml top layer containing the cells, 0.4% Bacto agar (Difco, Detroit,Mich.) with 10% FCS alone and with increasing concentrations of p75(0-132 pM), and gp30 (0-330 pM). All samples were run in triplicate andexperiments were carried out in FCS that had been tested for optimalcloning efficiency. Cells were incubated 7-9 days at 37° C. in 5% CO₂.Colonies larger than 60 μm were counted in a colony counter. Theexperiments were performed three times and the results werereproducible.

We observed that p75 at very low doses (3.3 pM, 0.25 ng/ml) had astimulatory growth effect on SK-Br-3 (FIG. 10). The proliferativeeffects of gp30 at equimolar concentrations were similar to those of p75(FIG. 10). As an additional control, EGF at concentrations from 1-100 nMhad no significant effects on the growth of SK-Br-3 cells. The resultsobtained with p75 argued against the possibility of an inhibitoryligand. Dose-related paradoxical effects of growth factors on cellularproliferation have been reported in the literature.

Low concentrations of gp30 stimulated the growth of SK-Br-3 cells, whilehigh concentrations were growth inhibitory (Lupu, et al., Science,249:1552 (1990)). EGFR-overexpressing breast cancer cells MDA-MB-468, aswell as the A431 cancer cells, are growth-inhibited by high doses of EGFbut are stimulated by very low doses of EGF (Kawamoto, et al., J. Biol.Chem., 249:7761 (1984); Ennis, et al., Molec. Endocr., 3:1830 (1989)).It has also been reported that cells overexpressing the estrogenreceptor are growth-inhibited by physiological doses of estrogen(Kushner, et al., Molec. Endocr., 4:1465 (1990)).

EXAMPLE 19 Interaction of p75 with p185^(erbB-2) Extracellular Domain

In additional experiments, we explored the interaction of p75 andp185^(erbB-2) soluble extracellular domain. SK-Br-3 cells were plated in35 mm tissue culture dishes (Costar, Cambridge, Mass.). A bottom layerof 1.0 ml IMEM (Biofluids) containing 0.6% agar and 10% FCS was preparedafter the bottom layer was solidified. The indicator cell, 10,000 cellsper dish were added in a 0.8 ml top layer containing the sample, 0.4%Bacto agar (Difco, Detroit, Mich.) and 10% FCS. The samples were p75(0.15 ng/ml), soluble recombinant ECD (12 μg/ml) and p75 in the presenceof EDC. All samples were run in triplicates and experiments were carriedout in FCS that had been tested for optimal cloning efficiency. Cellswere incubated 7-9 days at 37° C. in 5% CO₂. Colonies larger than 60 μmwere counted in a colony counter. The experiments were performed threetimes and the results were reproducible.

As might have been expected, addition of soluble p185^(erbB-2) ECD toSK-Br-3 cells inhibited their soft agar colony formation. The inhibitoryeffect of this ECD may be due either to dimerization of the solubledomain with the cellular p185^(erbB-2) receptor, or to binding andneutralization of p75, which is essential for their growth, thereforeleading to inhibition of cell proliferation. ECD inhibited colonyformation exclusively in those cells overexpressing p185^(erbB-2) (FIG.11). No inhibition was observed in MDA-MB-468 and MCF-7 cells. In orderto understand the mechanism by which erbB-2 ECD inhibited colonyformation of SK-Br-3 cells, growth-stimulatory doses of p75 were addedto ECD-treated cells. The inhibitory effect of the ECD was reversed bythe addition of stimulating doses of p75, suggesting that complexregulatory pathways may exist for p185^(erbB-2) (FIG. 11).

EXAMPLE 20 Large Scale Purification and Partial Amino Acid Sequence ofgp30

The following scheme of purification defines a preferred sequence ofsteps for gp30 recovery in different systems.

Heparin affinity chromatography: Media conditioned by MDA-MB-23 1 orSKBr-3 cells are clarified by centrifugation for 20 minutes at 2,000 rpmat 4° C. The supernatant is collected and stored at -70° C. Afterallowing heparin-sepharose beads to expand in PBS, 2 ml of gel beads areloaded on an Econo column and washed with about 100 bed volumes of PBS.Conditioned media are run through the beads by gravity (flow rate 20 to50 ml/hr). Gradient steps of 0.4M-2.0M NaCl are used until the 280nmabsorption during each step returns to baseline. Purification of 200liters of conditioned media was performed, and sufficient material wasobtained to perform a partial tryptic digestion.

Extraction of polypeptides from SDS-PAGE: After obtaining approximately5 μg of purified protein (gp30), extraction with acetonitrile wasperformed. After the extraction, amino acid analysis was determined toevaluate the amounts of protein in the sample. The purified material wassubjected to conventional N-terminal sequencing. After several trials itwas concluded that gp30 was N-terminally blocked.

Further amino acid sequencing was done by treating gp30 with trypsin(partial tryptic digestion). After the digestion, peptides wereseparated on HPLC on a C18-Reversed-phase column, single peptides werecollected as shown in FIG. 17A, and several peaks were subjected toprotein sequence analysis. Five independent fragments were sequenced asshown in FIG. 17B.

EXAMPLE 21 Isolation of a cDNA Clone That Encodes gp30

In order to allow identification of gp30-specific cDNA clones by nucleicacid hybridization, several oligonucleotides were designed based on theamino acid composition of the tryptic peptides. All the possible codonswere used in designing the shorter probe (16-48 combinations).End-labeled synthetic oligonucleotides were used to screen a cDNAlibrary that was prepared with mRNA isolated from MDA-MB-231 cells bystandard procedures. cDNA was synthesized with the superscript kit(Strategene). Column-fractionated double stranded cDNA was ligated intoan EcoRI site in a phage vector. Approximately three hundred thousandplaques were plated at a density of 30,000 plaques per 150 mm petridish. The screening procedure used Y1090 (r⁻) as the host bacteria.Areas containing plaques that resulted in positive hybridation wereselected and placed into a lambda diluent (10 mM Tri-pH:7.5, 10 mMMgCl₂). The phage lysate was used to reinfect Y1090 (r⁻) cells whichwere then replated at densities that allowed subsequent isolation of asingle positive plaque and the detection procedure was repeated. Toanalyze the positive clones, a phage DNA preparation was performed usinga mini preparation technique followed by a digestion with EcoRIrestriction enzyme, to get an estimate of the size of the cDNA insert.Initial sequencing reactions were performed using miniprep phage DNAwith lambda-zap forward and reverse primers (New England Biolabs).

Sixteen cDNA clones, which independently hybridized with both probes,were detected. After four screenings, 9 clones remained positive. Tofacilitate subsequent sequencing and further expression analysis, thecomplete cDNA inserts were amplified using the Polymerase Chain Reaction(PCR) technique with lambda-zap forward and reverse primers. The PCRamplified material was then subcloned into the EcoRI site of theTA-cloning vector (Invitrogen) and were sequenced using oligonucleotidescomplementary to the SP6 and T7 promotor binding sites as primers. Weobtained several positive clones. After extensive sequencing analysisand deduced amino acid sequence we determined the full length cDNA ofgp30 as shown in FIG. 18A. The sequence was computer-analyzed for thepresence of an eukaryotic secretory signal and the presence of theinitiation codons that confirm the Kozak consensus.

In addition to the full length cDNA clone shown in FIG. 18A that encodesgp30 (α1), three other cDNA's were obtained from the same expressionlibrary. These clones appear to be highly homologous to the cDNAsequence of gp30 (α1) but probably are alternatively spliced molecules.The size of these molecules are different from each other: α1: 2165oligonucleotides, β2: 1861 oligonucleotides, β3: 2260 oligonucleotidesand β4: 1681 oligonucleotides. The sequence of one of these clones(gp30/β1) is shown in FIG. 18B.

RNAse protection assay:

gp30 expression may be detected by RNAse protection assays, for which wehave generated several riboprobes (to the different forms of theoriginal gp30 cDNA). These riboprobes generate a different protectedsize that correlates with the gp30 cDNA, or its alternative splicedmolecules. Total RNA is hybridized with 120,000 cpm of labeled probe for12-16 hours at 50° C. Samples are then digested with 40 μg/ml of RNAse Aand 2 μg/ml of RNAse T1 for 30 min at 28° C. The RNAse digestion isterminated by the addition of both proteinase K 1 μg/ml and 1% SDS.Following one extraction with phenol/chloroform/isoamylalcohol(25:24:1), the samples are precipitated with 2 μg tRNA in absoluteethanol. Pellets are boiled in loading buffer, and the sample iselectrophoretically fractionated in 6% polyacrylamide gels containing 8Murea. The gels are dried and exposed at -70° C. to Kodak XAR5 filmbetween Chronex Quanta III intensifying screens.

We have generated a 432 bp riboprobe derived from the gp30 β1 sequence(605-1073) that can specifically protect the different forms of theligands. The probe contains the specific sequence for the β1 form. Asshown in FIG. 18C, using the β1 probe, four different fragments will beprotected: β1: 432 bp, α: 124 and 229 bp, β2: 178 and 229 bp and β3: 178bp. Expression of the erbB-2 ligands in breast cancer cell lines isshown in FIG. 18D.

EXAMPLE 22 Transfection of gp30 cDNA Into Host Cells

Full cDNA (approximately 2.4 kb) of the al form of gp30 was insertedinto a pCHC6 expression vector. Then the erbB-2 ligand α1 form wastransfected into a variety of breast cancer cells including MCF-7 cells.After collecting media from the stable transfected cells, we performederbB-2 phosphorylation assays using MDA-MB-453 cells as shown in FIG.19. MDA-MB-453 cells were grown to 90% confluence in 24-well plate andtreated at 37° C. with conditioned media from: untreated cells(-control); 100 μl of conditioned media×100 from MDA-MB-231 cells(+control); several batches of purified gp30 from conditioned mediaderived from MDA-MB-231 cells (heparin sepharose fractions); 100 μl ofmedia from different clones from the MCF-7/ligand transfected cells(clone A, B, C, D and E); 100 μl of media from the MCF/-7 wild type cellline (wt); and 100 μl of media derived from MCF-7/vector alone cells(wt-V). After 30 minutes media was removed and cells were lysed in 100μl of sample buffer. Proteins were then transferred to Hybond/ECLmembrane for immunoblotting with an anti-phosphotyrosine antibody (UBI)and developed by the ECL method (Amersham).

As can be seen, un-concentrated media from the stable transfected cellsinduced tyrosine phosphorylation of the erbB-2 receptor. Nophosphorylation was observed when conditioned media from MCF-7/WT orMCF-7/vector was used. Furthermore, we have shown that the secretedprotein can bind to heparin as well as the secreted form from MDA-MB-231cells. In conclusion, we have been able to construct a full length cDNAclone that translates into a biologically active ligand form.

EXAMPLE 23 Binding of gp30 to p185^(erbB-2)

The gp30 molecule may be purified using as a detection marker itsability to increase the level of tyrosine phosphorylation ofp185^(erbB-2) in living cells. The specificity of gp30 binding top185^(erbB-2) was demonstrated in several ways.

First, Scatchard analysis was performed using MDA-MB-453 cells andiodinated gp30. The incubation was performed in the presence ofincreasing concentrations of unlabelled gp30. Unbound ¹²⁵ I!-gp30 wasremoved by three consecutive washes with binding buffer (DMEM/F12 1:1and 0.1% BSA) and cells were solubilized in 0.1% SDS. Radioactivity wasdetermined by using a gama-counter. The analysis showed a single classof high affinity binding sites (Kd=112 pM) on the surface of the cells.

Second, to examine the presumed direct interaction between the purifiedfactor and p185^(erbB-2), the method of covalent cross-linking wasemployed. The isolated gp30 was radiolabeled with iodine and separatedfrom the free ¹²⁵ I by gel-filtration. The iodinated gp30 was incubatedwith MDA-MB-453 cells, and the bifunctional reagent ethylene glycolbis(EGS) was used to covalently cross-link gp30 to its receptor.

For the cross-linking experiments (FIG. 20), monolayers of MDA-MB-453cells (300,000 cell/well) were used. Control cultures received anunlabelled gp30 in a dose dependent manner (0.1-10 nM). A controlwithout cells, containing iodinated gp30 alone was performed asindicated (-) p185^(erbB-2). After 1 hr at 4° C. the cells weretransferred to incubation at room temperature and a chemicalcross-linker (EGS) was added to some plates as indicated. Cell lysateswere prepared after 45 minutes at 22° C. and subjected toimmunoprecipitation with an anti-erbB-2 C-terminal polyclonal antibodyand the precipitates were analyzed on a 6% SDS-PAGE. As shown in FIG.20A, the results of this analysis showed that cross-linking of iodinatedgp30 labeled one single band at an apparent molecular weight of 220 kDawhich was immunoprecipitated with a specific anti-erbB-2 antibody.

To determine the specificity of the cross-linking, unlabeled gp30 wasadded in a dose-dependent fashion, as shown in FIG. 20B. The resultssuggested that the 220 kDa protein is a 1:1 ratio complex of gp30 andp185^(erbB-2). In the absence of the cross-linker no protein wasdetected, thus excluding the possibility of a non-covalent nature ofinteraction.

EXAMPLE 24 Antibodies to gp30 Protein

Western blot analysis:

Western blots may be used to determine levels of gp30 protein. Severalanti-peptide polyclonal antibodies have been generated, and the abilityof those antibodies to recognize the ligand protein is shown in FIG. 21.Different concentrations of partially purified ligand were loaded into a4-20% SDS-PAGE, in a dose fashion (5, 10 and 20 ng/well). The gel wastransferred to the Hybond/ECL membrane. After transfer, the blot wasblocked using 5% BSA (RIA grade). The specific antibody was added(1:10000) for 1-2 hrs at RT. After the incubation with a secondantibody, the reaction was developed using the ECL method (Amersham). Nocross-reactivity was observed when pre-immune serum was used after eachtreatment. Using this technique, cells may be tested for the presence ofgp30 and the levels of gp30 in both conditioned media and cell lysatesmay be determined.

Two specific monoclonal anti-gp30 antibodies have been generated. Theseantibodies were developed by immunization of Balb/c mice with gp30protein.

Several hybridomas were identified and two were determined to bespecific to gp30. This was determined by using several syntheticpeptides derived from gp30 protein sequence. One of the monoclonalantibodies not only recognized gp30 protein but also recognized one ofthe synthetic peptides. The antibodies are specific for the erbB-2ligands and do not cross-react with TGFα, EGF, Amphiregulin and theHB-EGF.

EXAMPLE 25 Development of an ELISA Assay Using Anti-gp30 Antibodies

We have preliminary evidence that gp30 and erbB-2 can interact in aparacrine fashion. We have shown that fibroblasts derived from humanbreast tumors (stroma) express gp30. Therefore, we can postulate aparacrine loop.

Determination of the circulation levels of gp30 in breast cancerpatients is important for the confirmation of the paracrine loop. ELISAand RIA assays may be used for detection of gp30 in serum samples frombreast cancer patients. Both assays are a solid phase type assay (easyfor large screening).

An ELISA assay has been developed in which two different polyclonalantibodies are used, one of them being biotinylated. For this assay, a96 well plate is coated with one of the antibodies, and incubated withincreasing concentration of ligand as a standard curve and/or serialdilutions of serum samples from patients. A second anti-ligandbiotinylated antibody is used to determine the amount of ligand in eachsample. The assay is developed by streptavidin-horseradish peroxidase.

EXAMPLE 26 Immunohistochemical Staining for gp30 Using DifferentAnti-peptide Antibodies Termed α1, α2 and α3

FIG. 22 shows imaging analysis on confocal microscopy of immunostainingof breast cancer paraffin sections using anti-erbB-2 ligand antibodies:FIG. 22A was immunostained with a polyclonal α1 antibody (Affinitypurified antibody 1 μg/ml). FIG. 22B was immunostained with a monoclonalantibody (7B3-strait hybridoma supernatant. The antibody used in FIG.22D was monoclonal antibody (10F10)-strait hybridoma supernatant. Theslide shown in FIG. 22E was stained using 10F10 antibody in the presenceof a blocking peptide (1 μg/ml). FIGS. 22C and F represents phasestaining. An anti-rabbit or anti-mouse FITC conjugated second antibodywas used to develop the staining.

As shown in FIG. 22, we have been able to obtain positive staining usingseveral of the anti-ligand antibodies. Furthermore, we have shown thatthe staining was specific to ligand, since it was completely abolishedby the addition of the respective peptides. Staining was observedintracellular in breast cancer epithelial cells and also in someadjacent fibroblasts.

EXAMPLE 27 In situ Hybridization to Detect gp30 Expression

In situ hybridization to gp30 mRNA is performed according to previouslydescribed protocols developed for other growth factors, using differentriboprobes as described above to determine the expression of gp30 and/orthe alternative spliced molecules. In brief, a pretreatment step for aparaffin embedded section is performed. All pre-ribonuclease washingsteps are done with autoclaved polypropylene staining dishes. Sectionsare deparaffinized and rehydrated through alcohol series. Basic proteinsare removed by incubation for 20 minutes in 0.2M HCl. Sections aretreated with proteinase K and then incubated with glycine, washed andthen acetylated in fresh acetic anhydride. Sections are washed anddehydrated through ethanol. Hybridization mix is added containing 5×10⁷cpm/ml of probe. Washes are performed and sections are exposed to NBT-2emulsion for 1 to 3 weeks. After fixation sections are stained withhematoxylin-eosin and examined under the microscope.

EXAMPLE 28 RT-PCR from mRNA Extracted From Paraffin-embedded HumanTissues

In order to determine the level of gp30 expression and to comparebetween the different forms of the erbB-2 ligands (alternatively splicedmolecules), PCR may be performed on RNA isolated from fixed sections. Ithas been previously shown that RNA extracted from formalin-fixed andparaffin embedded tissues can be reproducibly used as a substrate forPCR amplification. The hospital archives of human pathological tissuesof biopsy or surgical origin are thus amenable to the analysis ofexpressed gp30.

In brief, RNA is prepared from the embedded tissue and cDNA fragmentsgenerated using reverse transcriptase and random hexamers. Specificprimers covering a fragments of 900 nucleotides from the 5' and 3' endof gp30 cDNA may be used to search for specific cDNA by 30-35 rounds ofPCR amplification. Southern blotting of selected negative and positivesamples may be used to monitor the sensitivity of the assay.

PCR analysis:

Cells are treated and RNA is isolated. Primers located in the 5' and the3' end of the cDNA are used, depending upon the sequence of interest.Sets of primers that can amplify fragments from 1.3 kb to 400 bp caneasily be designed by the skilled worker from the sequences disclosedherein. Preferably, these sets cover the open reading frame of theactive site of gp30 (the EGF-like domain). In addition some of thecombinations may include the Immunoglobulin domain and the transmembranedomain. A test for specificity may be performed by cutting the productwith a restriction enzyme that should yield two fragments of known size.XhoI will serve as a restriction enzyme for this purpose, since it is aunique restriction site present in all combinations of primers for gp30.

The primers shown in FIG. 23A have been derived from the α1 sequence andused to determine gp30 expression based on amplification of the sequenceshown in FIG. 23B. A control is necessary to validate the results. Aunique XhoI restriction site (shown in the sequence in FIG. 23B) thatwill generate two distinctive fragments from the product amplified withall the possible combinations of primers makes it possible to determinethat the amplified fragment represents the specific gene of interest(gp30). An example of the results obtained are shown in FIG. 23C.

FIG. 23C shows amplification of a specific ligand PCR product from 20 μparaffin section from breast cancer cell lines. MCF-7 cells (A=negativecontrol) and MDA-MB-231 cells (B=positive control), were used todetermine the conditions of the isolation and the RT-PCR. After theisolation, RT-PCR was performed using two different sets of primers asdescribed earlier. As can be seen in FIG. 23C (left panel),amplification using specific β-actin probes (C) a band at 650 bp wasobtained from both extractions, whereas with the erbB-2 ligand primers(D), amplification was seen at 900 bp only from the mRNA derived fromthe MDA-MB-231 cells. In addition, mRNA was isolated from differentbreast cancer tumor samples as shown in FIG. 23C (right panel), and aspecific 900 bp fragment was amplified from a tumor sample (E) that wasdetermined by RNAse protection assay to be positive. In order todetermine that the amplified fragment was the gene of interest, arestriction enzyme digestion using Xho Iwas performed, and two expectedfragments were generated (F), showing the specificity of the assay.

EXAMPLE 29 Modulation of the Invasive Phenotype of Breast Cancer Cells

The constitutive effect of erbB-2 overexpression on human breast cancercell invasiveness was examined for possible contribution to the poorerprognosis associated with this factor. erbB-2 positive cell lines(SKBR-3, BT-474 and MDA-MB-453) are all vimentin-negative, andaccordingly show low constitutive invasiveness in the Boyden chamberassays for chemoinvasion and chemotaxis toward fibroblast conditionedmedia, as well as the Matrigel outgrowth assay. When these cells weretreated with low concentrations of gp30, it was observed that inductionof chemotaxis and chemoinvasion occurred in a dose-dependent mannerwhich is similar to the growth response. FIG. 24 shows this effect forSKBR-3 cells. Cells were treated in the chamber with increasingconcentrations of gp30, and incubated for 16 hours. Data pointsrepresent the mean±SEM from triplicate filters in a representativeexperiment. Similar dose response curves have been obtained on at leasttwo other occasions. gp30 effects on chemoinvasion and chemomigrationwere seen to various extents in the SKBR-3 and MDA-MB-453 cells, all ofwhich contain erbB-2 receptors, the latter lacking measurableEGF-receptor. The specificity of this effect to the presence of gp30 wasascertained by blocking the effect in SKBr-3 cells with the addition ofexcess soluble erbB-2-ECD (data not shown).

EXAMPLE 30 The Proliferation of BT-474 Cells is Regulated by17β-estradiol and the erbB-2 Ligand gp30

A significant fraction of breast cancer cells exhibit simultaneousexpression of estrogen receptor (ER) and erbB-2. It has been reportedthat 17β-estradiol induces down-regulation of erbB-2 in such cells. Inthis example, we studied the interaction between these two receptors intumor progression. As a model we used two cell lines: BT-474 andMDA-MB-361 cells.

In vitro growth effects of 17β-estradiol

To determine the effect of 17β-estradiol, anchorage-dependent growthassays were preformed using medium containing serum as control (fetalcalf serum, FCS) or phenol red-free hormone-depleted serum(charcoal-stripped calf serum, CCS), with or without the addition of a10⁻⁹ M of 17β-estradiol. The depletion of hormones from the culturemedium resulted in markedly slower cell proliferation, with BT-474 cellsundergoing less than 1 doubling after 12 days. When 17β-estradiol wasadded to the medium the same proliferation rate as cells cultured withunprocessed serum was observed. Similarly, when a serum-free mediumcontaining insulin, transferrin and selenium (HL-1, Ventrex) was used,BT-474 cells did not proliferate, but upon the addition of 17β-estradiol(10⁻⁹ M), the same doubling times as above were observed (data notshown). Similar results were obtained when MDA-MB-361 cells were used;their proliferation was markedly reduced by hormonal deprivation, andthe addition of 17β-estradiol induced a dramatic proliferative response.In addition the growth of these cells was modulated when gp30 was used,as previously shown for SKBr-3 cells.

ER expression in BT-474 cells modulated by erbB-2 ligand:

The effects of estradiol and gp30 on the expression of estrogen receptorand progesterone receptor (PgR) were studied in BT-474 cells. BT-474cells were grown in IMEM containing 5% FCS. When the cells were 70-80%confluent, the media was changed to IMEM (phenol red-free) supplementedwith 5% CCS for two days. Cells were treated for 24 hours with 10⁻⁹ Mestradiol, 0.02 to 2.0 ng/ml gp30, or 0.04 to 4.0 ng/ml p75. ER and PgRprotein were assayed using enzyme immunoassay (Abbot Laboratories).

As shown in FIG. 25, treatment of cells with 10⁻⁹ M estradiol for 24hours resulted in a decline in ER protein of approximately 80% from alevel of 35 fmol/mg protein in control cells to 6 fmol/mg protein intreated cells (left panel, 0 ng/ml gp30). Treatment of cells withestradiol resulted in 2.5-fold increase in PgR (right panel, 0 ng/mlgp30). When cells were treated with concentrations of gp30 from 0.02 to2.0 ng/ml for 24 hours there was a concentration dependent decrease inthe level of ER. The higher concentration of gp30 resulted in a decreasein ER to approximately 20% of control values. gp30 had no effect on thelevel of PgR. When estradiol and gp30 were added simultaneously tocells, gp30 at low concentrations blocked the regulation of ER byestradiol. All concentrations of gp30 blocked estradiol induction ofPgR.

Similar results were obtained when MCF-7 and/or MDA-MB-361 cells wereused. Furthermore, identical results to those for protein determination,were obtained when mRNA levels were assay by an RNAse protection assay.

erbB-2 receptor expression in BT-474 cells as affected by 17 β estradioland gp30:

The levels of erbB-2 protein were determined using a radio-receptorassay which employs an iodinated antibody that binds to theextracellular domain of erbB-2 (¹²⁵ I-4D5 antibody).

BT-474 cells were plated (20,000 cells/well) in 24 well plates in IMEM(phenol red-free) supplemented with 5% FCS. After 24 hrs the media wasremoved and replaced with media supplemented with 5% CCS with or withoutthe addition of 17β-estradiol 10⁻⁹. After 48 hrs media were replacedwith IMEM (phenol-red-free) supplemented with 5% CCS, in the presence orabsence of increasing concentrations of gp30 (0.02, 0.2, 2.0 ng/ml) orp75 (0.04, 0.4, 4.0 ng/ml) and EGF (1.0 ng/ml) for additional 24 hrs.The erbB-2 protein levels were assayed using a competition binding assayusing (¹²⁵ I-4D5 or ¹²⁵ I-6E9) anti-erbB-2 antibodies against theextracellular domain.

Typical results are shown in FIG. 26. Overnight treatment of BT-474cells with 10⁻⁹ M 17β-estradiol, resulted in a decrease in antibodybinding to erbB-2 of approximately 45%. 16 hrs treatment with gp30 (2ng/ml) in hormone-depleted medium did not noticeably change the levelsof erbB-2. However, this concentration of gp30 completely abrogated thedown-regulating effect induced by 17β-estradiol (FIG. 26). gp30concentrations as low as 0.02 ng/ml were sufficient to block the effectof 17β-estradiol. Epidermal growth factor (1 ng/ml) did not alter thelevels of erbB-2 and, in contrast to gp30, did not interfere with theeffect of 17β-estradiol on erbB-2 levels.

Similar results were obtained when MDA-MB-361 and/or MCF-7 cells wereused. We have also shown by RNAse protection assays, that modulation atthe mRNA level is similar to the modulation of the protein level.

EXAMPLE 31 Time Course of erbB-2 Down-regulation After Estrogen Additionand Blockage by gp30

The level of the erbB-2 mRNA in gp30/estrogen treated cells, versusuntreated cells and estrogen depleted cells may be determined over time.Estrogen is added for a length of time and the mRNA levels determined byNorthern blot analysis. The difference observed after 72 hours should beenough to distinguish half-life changes, but shorter times of treatmentmay also be possible. Actinomycin D may be added to a concentration thathas previously been shown to result in greater than 90% inhibition of ³H!uridine incorporation in TCA precipitable material within 1 hour. Theprecise dose may be determined by the skilled worker, but initially adose of 5 μg/ml may be used. Total RNA is extracted at various intervalsafter the addition of Actinomycin D and subjected to Northern analysis,using fragments of the erbB-2 oncogene coding sequence as probes.Hybridization may be quantified by densitometry.

When estrogen was added to cultured cells like BT-474 cells,down-regulation at the mRNA level was observed at the first time pointtested, i.e. 72 hrs after the addition of estrogen. An effect on erbB-2mRNA level may be observed at an earlier time period after estrogenaddition, and detection of down-regulation can be used to determine theoptimal time and dose for gp30 effect. As a control for mRNA loading onemay use both the GAPDH gene, which is a nonestrogen-induced transcript,and a pS2 probe which is an estrogen-induced transcript.

Regulation erbB-2 expression after treatment with gp30/estrogen andanti-estrogens may also be investigated by performing nuclear-run-onassays. These assays will detect expression at the mRNA level. Thenuclear transcription run-on assay may be performed as follows. Isolatednuclei are incubated with ³² P-UTP and unlabeled ATP, CTP, and GTP. Theradiolabeled RNA transcripts are isolated and hybridized to an excess ofdenatured plasmid DNA immobilized on a nitrocellulose filter. Thedenatured plasmid used for the detection of specific transcripts willencode erbB-2 receptor, GAPDH and pS2. Autoradiography is analyzed bydensitometry and the results are normalized for the number of nuclei orby comparison to the transcriptional level of GAPDH.

SUMMARY

In brief, we have identified a novel polypeptide of 75 kDa that binds tothe p185^(erbB-2) receptor. The effects of p75 on cells with very highlevels of erbB-2 were similar to the reported effects of the otherligand, gp30. In contrast to gp30, p75 appears to be specific forp185^(erbB-2) receptor. Furthermore, we have provided evidence thatcells that overexpress the erbB-2 receptor may also secrete one of itsligands, which is required for their proliferation, therefore implyingan autocrine loop. We believe that manipulation of this and other erbB-2ligands may turn out to have an important biological effect on growth ofhuman neoplasia.

Modifications of the above-described modes for carrying out theinvention that are obvious to persons of skill in medicine, immunology,hybridoma technology, pharmacology, and/or related fields are intendedto be within the scope of the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Having now described the invention, it will now be apparent to one ofordinary skill in the art that many changes and modifications can bemade to the above embodiments without departing from the scope andspirit of the present invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 18    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    LysGlyLysGlyLysLysXaaGluArgGlyArgGlyLysLysProGly    151015    SerAlaAlaXaaProGlnSerProAlaLeuPro    2025    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    LeuValLeuArgCysGluThrSerSerThrTyrSerSerLeuAlaPhe    151015    LysTrpPheLysAsnGly    20    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    LeuGlyAsnAspSerAlaSerAlaAsnIleThrIleValGluSerAsn    151015    GluIleIleThrGlyAsnMetProAla    2025    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    AspSerGlyGluTyrMetCysLysValIleSer    1510    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    LeuValLysCysAlaGluLysGluLysThrPheCysValAsnGlyGly    151015    GluCysPheMetValLysAsp    20    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2164 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CCGATCCGAGCCCTTGGACCAAACTCGCCTGCGCCGAGAGCCGTCCGCGTAGAGCGCTCC60    GTCTCCGGCGAGATGTCCGAGCGCAAAGAAGGCAGAGGCAAAGGGAAGGGCAAGAAGAAG120    GAGCGAGGCTCCGGCAAGAAGCCGGAGTCCGCGGCGGGCAGCCAGAGCCCAGCCTTGCCT180    CCCCGATTGAAAGAGATGAAAAGCCAGGAATCGGCTGCAGGTTCCAAACTAGTCCTTCGG240    TGTGAAACCAGTTCTGAATACTCCTCTCTCAGATTCAAGTGGTTCAAGAATGGGAATGAA300    TTGAATCGAAAAAACAAACCACAAAATATCAAGATACAAAAAAAGCCAGGGAAGTCAGAA360    CTTCGCATTAACAAAGCATCACTGGCTGATTCTGGAGAGTATATGTGCAAAGTGATCAGC420    AAATTAGGAAATGACAGTGCCTCTGCCAATATCACCATCGTGGAATCAAACGAGATCATC480    ACTGGTATGCCAGCCTCAACTGAAGGAGCATATGTGTCTTCAGAGTCTCCCATTAGAATA540    TCAGTATCCACAGAAGGAGCAAATACTTCTTCATCTACATCTACATCCACCACTGGGACA600    AGCCATCTTGTAAAATGTGCGGAGAAGGAGAAAACTTTCTGTGTGAATGGAGGGGAGTGC660    TTCATGGTGAAAGACCTTTCAAACCCCTCGAGATACTTGTGCAAGTGCCAACCTGGATTC720    ACTGGAGCAAGATGTACTGAGAATGTGCCCATGAAAGTCCAAAACCAAGAAAAGGCGGAG780    GAGCTGTACCAGAAGAGAGTGCTGACCATAACCGGCATCTGCATCGCCCTCCTTGTGGTC840    GGCATCATGTGTGTGGTGGCCTACTGCAAAACCAAGAAACAGCGGAAAAAGCTGCATGAC900    CGTCTTCGGCAGAGCCTTCGGTCTGAACGAAACAATATGATGAACATTGCCAATGGGCCT960    CACCATCCTAACCCACCCCCCGAGAATGTCCAGCTGGTGAATCAATACGTATCTAAAAAC1020    GTCATCTCCAGTGAGCATATTGTTGAGAGAGAAGCAGAGACATCCTTTTCCACCAGTCAC1080    TATACTTCCACAGCCCATCACTCCACTACTGTCACCCAGACTCCTAGCCACAGCTGGAGC1140    AACGGACACACTGAAAGCATCCTTTCCGAAAGCCACTCTGTAATCGTGATGTCATCCGTA1200    GAAAACAGTAGGCACAGCAGCCCAACTGGGGGCCCAAGAGGACGTCTTAATGGCACAGGA1260    GGCCCTCGTGAATGTAACAGCTTCCTCAGGCATGCCAGAGAAACCCCTGATTCCTACCGA1320    GACTCTCCTCATAGTGAAAGGTATGTGTCAGCCATGACCACCCCGGCTCGTATGTCACCT1380    GTAGATTTCCACACGCCAAGCTCCCCCAAATCGCCCCCTTCGGAAATGTCTCCACCCGTG1440    TCCAGCATGACGGTGTCCATGCCTTCCATGGCGGTCAGCCCCTTCATGGAAGAAGAGAGA1500    CCTCTACTTCTCGTGACACCACCAAGGCTGCGGGAGAAGAAGTTTGACCATCACCCTCAG1560    CAGTTCAGCTCCTTCCACCACAACCCCGCGCATGACAGTAACAGCCTCCCTGCTAGCCCC1620    TTGAGGATAGTGGAGGATGAGGAGTATGAAACGACCCAAGAGTACGAGCCAGCCCAAGAG1680    CCTGTTAAGAAACTCGCCAATAGCCGGCGGGCCAAAAGAACCAAGCCCAATGGCCACATT1740    GCTAACAGATTGGAAGTGGACAGCAACACAAGCTCCCAGAGCAGTAACTCAGAGAGTGAA1800    ACAGAAGATGAAAGAGTAGGTGAAGATACGCCTTTCCTGGGCATACAGAACCCCCTGGCA1860    GCCAGTCTTGAGGCAACACCTGCCTTCCGCCTGGCTGACAGCAGGACTAACCCAGCAGGC1920    CGCTTCTCGACACAGGAAGAAATCCAGGCCAGGCTGTCTAGTGTAATTGCTAACCAAGAC1980    CCTATTGCTGTATAAAACCTAAATAAACACATAGATTCACCTGTAAAACTTTATTTTATA2040    TAATAAAGTATTCCACCTTAAATTAAACAATTTATTTTATTTTAGCAGTTCTGCAAATAG2100    AAAACAGGAAAAAAACTTTTATAAATTAAATATATGTATGTAAAAATGAAAAAAAAAAAA2160    AAAA2164    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2199 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GGGACAAACTTTTCCCAAACCCGATCCGAGCCCTTGGACCAAACTCGCCTGCGCCGAGAG60    CCGTCCGCGTAGAGCGCTCCGTCTCCGGCGAGATGTCCGAGCGCAAAGAAGGCAGAGGCA120    AAGGGAAGGGCAAGAAGAAGGAGCGAGGCTCCGGCAAGAAGCCGGAGTCCGCGGCGGGCA180    GCCAGAGCCCAGCCTTGCCTCCCCAATTGAAAGAGATGAAAAGCCAGGAATCGGCTGCAG240    GTTCCAAACTAGTCCTTCGGTGTGAAACCAGTTCTGAATACTCCTCTCTCAGATTCAAGT300    GGTTCAAGAATGGGAATGAATTGAATCGAAAAAACAAACCACAAAATATCAAGATACAAA360    AAAAGCCAGGGAAGTCAGAACTTCGCATTAACAAAGCATCACTGGCTGATTCTGGAGAGT420    ATATGTGCAAAGTGATCAGCAAATTAGGAAATGACAGTGCCTCTGCCAATATCACCATCG480    TGGAATCAAACGAGATCATCACTGGTATGCCAGCCTCAACTGAAGGAGCATATGTGTCTT540    CAGAGTCTCCCATTAGAATATCAGTATCCACAGAAGGAGCAAATACTTCTTCATCTACAT600    CTACATCCACCACTGGGACAAGCCATCTTGTAAAATGTGCGGAGAAGGAGAAAACTTTCT660    GTGTGAATGGAGGGGAGTGCTTCATGGTGAAAGACCTTTCAAACCCCTCGAGATACTTGT720    GCAAGTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAACTACGTAATGGCCAGCTTCT780    ACAAGCATCTTGGGATTGAATTTATGGAGGCGGAGGAGCTGTACCAGAAGAGAGTGCTGA840    CCATAACCGGCATCTGCATCGCCCTCCTTGTGGTCGGCATCATGTGTGTGGTGGCCTACT900    GCAAAACCAAGAAACAGCGGAAAAAGCTGCATGACCGTCTTCGGCAGAGCCTTCGGTCTG960    AACGAAACAATATGATGAACATTGCCAATGGGCCTCACCATCCTAACCCACCCCCCGAGA1020    ATGTCCAGCTGGTGAATCAATACGTATCTAAAAACGTCATCTCCAGTGAGCATATTGTTG1080    AGAGAGAAGCAGAGACATCCTTTTCCACCAGTCACTATACTTCCACAGCCCATCACTCCA1140    CTACTGTCACCCAGACTCCTAGCCACAGCTGGAGCAACGGACACACTGAAAGCATCCTTT1200    CCGAAAGCCACTCTGTAATCGTGATGTCATCCGTAGAAAACAGTAGGCACAGCAGCCCAA1260    CTGGGGGCCCAAGAGGACGTCTTAATGGCACAGGAGGCCCTCGTGAATGTAACAGCTTCC1320    TCAGGCATGCCAGAGAAACCCCTGATTCCTACCGAGACTCTCCTCATAGTGAAAGGTATG1380    TGTCAGCCATGACCACCCCGGCTCGTATGTCACCTGTAGATTTCCACACGCCAAGCTCCC1440    CCAAATCGCCCCCTTCGGAAATGTCTCCACCCGTGTCCAGCATGACGGTGTCCATGCCTT1500    CCATGGCGGTCAGCCCCTTCATGGAAGAAGAGAGACCTCTACTTCTCGTGACACCACCAA1560    GGCTGCGGGAGAAGAAGTTTGACCATCACCCTCAGCAGTTCAGCTCCTTCCACCACAACC1620    CCGCGCATGACAGTAACAGCCTCCCTGCTAGCCCCTTGAGGATAGTGGAGGATGAGGAGT1680    ATGAAACGACCCAAGAGTACGAGCCAGCCCAAGAGCCTGTTAAGAAACTCGCCAATAGCC1740    GGCGGGCCAAAAGAACCAAGCCCAATGGCCACATTGCTAACAGATTGGAAGTGGACAGCA1800    ACACAAGCTCCCAGAGCAGTAACTCAGAGAGTGAAACAGAAGATGAAAGAGTAGGTGAAG1860    ATACGCCTTTCCTGGGCATACAGAACCCCCTGGCAGCCAGTCTTGAGGCAACACCTGCCT1920    TCCGCCTGGCTGACAGCAGGACTAACCCAGCAGGCCGCTTCTCGACACAGGAAGAAATCC1980    AGGCCAGGCTGTCTAGTGTAATTGCTAACCAAGACCCTATTGCTGTATAAAACCTAAATA2040    AACACATAGATTCACCTGTAAAACTTTATTTTATATAATAAAGTATTCCACCTTAAATTA2100    AACAATTTATTTTATTTTAGCAGTTCTGCAAATAGAAAACAGGAAAAAAACTTTTATAAA2160    TTAAATATATGTATGTAAAAATGAAAAAAAAAAAAAAAA2199    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GAATGTGCCCATGAAAGTCCAAAACCA27    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    AGAAAAGGCGGAGGAGCT18    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 61 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 49..61    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..24    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    CTACGTAATGGCCCAGCTTCTACAAGCATCTTGGGATTGAATTTATGGAGGCGGAGGAGC60    T61    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..18    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    AARGGNAARGGNAARAARNN20    LysGlyLysGlyLysLys    15    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    LysGlyLysGlyLysLys    15    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    TTYCCNTTYCCNTTYTTYNN20    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..18    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GGNGARUAYAUGUGYAARGU20    GlyGluTyrMetCysLysVal    15    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GlyGluTyrMetCysLysVal    15    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    CCNCTYATRTACACRTTYCA20    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 483 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..483    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    AAAGGGAAGGGCAAGAAGAAGGAGCGAGGCTCCGGCAAGAAGCCGGAG48    LysGlyLysGlyLysLysLysGluArgGlySerGlyLysLysProGlu    151015    TCCGCGGCGGGCAGCCAGAGCCCAGCCTTGCCTCCCCGATTGAAAGAG96    SerAlaAlaGlySerGlnSerProAlaLeuProProArgLeuLysGlu    202530    ATGAAAAGCCAGGAATCGGCTGCAGGTTCCAAACTAGTCCTTCGGTGT144    MetLysSerGlnGluSerAlaAlaGlySerLysLeuValLeuArgCys    354045    GAAACCAGTTCTGAATACTCCTCTCTCAGATTCAAGTGGTTCAAGAAT192    GluThrSerSerGluTyrSerSerLeuArgPheLysTrpPheLysAsn    505560    GGGAATGAATTGAATCGAAAAAACAAACCACAAAATATCAAGATACAA240    GlyAsnGluLeuAsnArgLysAsnLysProGlnAsnIleLysIleGln    65707580    AAAAAGCCAGGGAAGTCAGAACTTCGCATTAACAAAGCATCACTGGCT288    LysLysProGlyLysSerGluLeuArgIleAsnLysAlaSerLeuAla    859095    GATTCTGGAGAGTATATGTGCAAAGTGATCAGCAAATTAGGAAATGAC336    AspSerGlyGluTyrMetCysLysValIleSerLysLeuGlyAsnAsp    100105110    AGTGCCTCTGCCAATATCACCATCGTGGAATCAAACGAGATCATCACT384    SerAlaSerAlaAsnIleThrIleValGluSerAsnGluIleIleThr    115120125    GGTATGCCAGCCTCAACTGAAGGAGCATATGTGTCTTCAGAGTCTCCC432    GlyMetProAlaSerThrGluGlyAlaTyrValSerSerGluSerPro    130135140    ATTAGAATATCAGTATCCACAGAAGGAGAGTATATGTGCAAAGTGATC480    IleArgIleSerValSerThrGluGlyGluTyrMetCysLysValIle    145150155160    AGC483    Ser    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 161 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    LysGlyLysGlyLysLysLysGluArgGlySerGlyLysLysProGlu    151015    SerAlaAlaGlySerGlnSerProAlaLeuProProArgLeuLysGlu    202530    MetLysSerGlnGluSerAlaAlaGlySerLysLeuValLeuArgCys    354045    GluThrSerSerGluTyrSerSerLeuArgPheLysTrpPheLysAsn    505560    GlyAsnGluLeuAsnArgLysAsnLysProGlnAsnIleLysIleGln    65707580    LysLysProGlyLysSerGluLeuArgIleAsnLysAlaSerLeuAla    859095    AspSerGlyGluTyrMetCysLysValIleSerLysLeuGlyAsnAsp    100105110    SerAlaSerAlaAsnIleThrIleValGluSerAsnGluIleIleThr    115120125    GlyMetProAlaSerThrGluGlyAlaTyrValSerSerGluSerPro    130135140    IleArgIleSerValSerThrGluGlyGluTyrMetCysLysValIle    145150155160    Ser    __________________________________________________________________________

We claim:
 1. An antibody which specifically binds a substantially pureprotein, wherein said protein:binds to heparin sepharose; has apparentmolecular weight as measured by SDS PAGE of about 30 kDa; has apparentmolecular weight as measured by SDS PAGE of about 22 kDa afterN-glycanase digestion; has an amino acid sequence of a peptide ofapproximately 22 kDa molecular weight as measured by SDS PAGE producedby in vitro translation of poly A RNA from MDA-MB-231 cells; uponhydrolysis with S. aureus V8 protease or elastase produces a peptidepattern the same as that produced by the same protease from a peptide ofapproximately 22 kDa molecular weight as measured by SDS PAGE producedby in vitro translation of poly A RNA from MDA-MB-231 cells; and inducesphosphorylation of p185^(erbB-2) in cells that overexpress erbB-2;induces internalization of the erbB-2 receptor; stimulates growth ofcells which overexpress erbB-2; inhibits growth of cells thatoverexpress erbB-2; reverses Mab 4D5-dependent inhibition of erbB-2overexpressing cells; induces differentiation of erbB-2 overexpressingcells; and stimulates phosphorylation of epidermal growth factorreceptor (EGFR) in EGFR expressing cells wherein said antibody does notcross react with TGFα,EGF, Amphiregulin and the HB-EGF.
 2. The antibodyof claim 1 wherein the substantially pure protein has an amino acidsequence which corresponds to an amino acid sequence encoded by the DNAsequence of FIG. 18A as shown in SEQ ID NO: 6, FIG. 18B as shown in SEQID NO: 7 and, FIG. 23B as shown in SEQ ID NO:
 17. 3. The antibody ofclaim 1 is a polyclonal antibody.
 4. The antibody of claim 1 is amonoclonal antibody.
 5. The antibody of claim 2 is a polyclonalantibody.
 6. The antibody of claim 2 is a monoclonal antibody.